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Cell Cycle and Checkpoint Control
Summary: Helen Piwnica-Worms is interested in how the cell division cycle is regulated and how perturbations in cell cycle control contribute to human cancer.
Our goals include delineating how the cell division cycle is regulated, determining how cancer cells derail cell cycle regulatory pathways, and ultimately using this information to treat human cancers. The cell division cycle replicates the DNA with fidelity once during S phase and ensures that identical chromosomal copies are segregated equally to the two daughter cells during mitosis. Complexes consisting of cyclins and cyclin-dependent protein kinases (CDKs) regulate progression through the cell cycle. In addition, checkpoints exist to monitor the integrity and replication status of the genetic material before cells commit to either replicate (in S phase) or segregate (in mitosis) their DNA. These checkpoints ultimately interface with the cell cycle machinery to block CDK activation and cell cycle progression when appropriate. Checkpoints ensure the accurate reproduction and dispersion of the cell's genetic material, while defects in checkpoint pathways contribute to the onset and progression of human cancers.
CDC25 Phosphatases
The CDC25 phosphatases positively regulate cell division by activating CDKs. Yeast encode a single Cdc25 protein, whereas humans and mice encode three family members (CDC25A, -B, and -C). We are interested in determining the contributions made by individual family members to embryonic and adult cell cycles in mammals. We generated mice disrupted for Cdc25C and found, unexpectedly, that they are healthy. Cells derived from these mice exhibit normal cell cycles and checkpoint responses. Cdc25B-null mice are also normal (although females are sterile), and cells derived from these mice have normal cell cycles.
We then generated double mutants and obtained mice lacking both Cdc25B and Cdc25C at the expected Mendelian ratios. These findings indicate that CDC25A is able to compensate functionally for loss of CDC25B and CDC25C in mice and that CDC25A can regulate all cell cycle transitions. To test this, we employed gene-targeting strategies to disrupt Cdc25A alone and in combination with Cdc25B and Cdc25C in mice. These studies uncovered an essential role for CDC25A during early embryogenesis, demonstrated that CDC25A is not essential in adult animals, and demonstrated that simultaneous deletion of all three CDC25 family members in adults results in rapid death due to a severe loss of absorptive surface villi throughout the small intestine. This model also provides a platform to disrupt cell division selectively in any tissue of the mouse. As an example, we chose to explore how small intestinal epithelial stem cells and progenitors respond to acute disruption of cell division, and we intend to investigate responses of other tissues as well. This system is being explored as a model to identify the small intestinal epithelial stem cell. We are also using this model to compare how small intestinal epithelial cells respond to acute disruption of cell division through CDC25 loss versus through chemotherapy treatment. This is providing insight into how patients may respond to treatments involving CDC25 inhibitors.
Checkpoint Control
Checkpoints induce cell cycle arrest or apoptosis in response to genotoxic or replication stress, while defects in checkpoints result in genomic instability and cancer predisposition. The ATM and ATR protein kinases are critical components of checkpoints that phosphorylate a plethora of cellular proteins to regulate DNA repair, cell death, and cell cycle progression. Downstream of ATM and ATR are two structurally unrelated protein kinases, Chk1 and Chk2. Chk2 is regulated by ATM in cells with double-strand DNA breaks, and Chk1 is regulated by ATR in cells experiencing replication stress. ATM and Chk2 are mutated in certain hereditary cancers. Following DNA damage, Chk2 oligomerizes in a phosphorylation-dependent manner, a process that ultimately results in its full activation. Previously we demonstrated that Chk2 oligomerizes and auto- or transphosphorylates in the absence of exogenous DNA damage when expressed at high levels in either mammalian cells or in bacteria. In collaboration with Reid Townsend (Proteomics Center, Washington University School of Medicine), we carried out a comprehensive phosphorylation analysis of bacterially produced Chk2 and identified several novel autophosphorylation sites. One of these sites was subsequently shown to be required for Chk2 ubiquitination and effector function.
Another goal of our laboratory is to understand how checkpoints interface with cell cycle regulators to prevent activation of CDKs during checkpoint responses. We demonstrated that CDC25A and CDC25C are key targets of negative regulation by checkpoints, that Chk1 is a major regulator of CDC25A, and that the integrity of the Chk1-CDC25A regulatory pathway is required for cells to delay in the S and G2 phases of the cell cycle in response to DNA damage. We uncovered an ATR-Chk1-PP2A (protein phosphatase 2A) regulatory circuit that keeps Chk1 in a low-activity state during an unperturbed cell division cycle but at the same time keeps Chk1 poised to respond rapidly in the event that cells encounter DNA damage. In addition, we identified GSK-3β (glycogen synthase kinase-3β) as a novel regulator of CDC25A in early cell cycle phases and uncovered a molecular mechanism that may account for the high frequency of CDC25A overproduction in human cancers.
Molecular Imaging Studies
CDC25A has been difficult to study in vivo because of its low level of expression and the lack of adequate probes with which to detect it. Most of the mechanistic insights that we and others have had regarding CDC25A regulation have come from studies in cultured cells. We believe it is critical to verify that the CDC25A-regulatory pathways operate in primary cells as well as in vertebrate animals. We are particularly interested in the Chk1-CDC25A pathway, as we have designed phase 1 clinical trials based on targeting this pathway in cancer. Therefore, we are developing molecular imaging technologies that enable imaging of endogenous CDC25A in intact cells and in mice. We generated knock-in mice expressing a fusion protein between CDC25A and click beetle red luciferase from the Cdc25A locus. These mice enable noninvasive and repetitive imaging of endogenous CDC25A in mice under steady-state conditions and in response to DNA-damaging agents and drugs that target cell cycle checkpoints. We are using these mice to model an ongoing phase 1 clinical trial. To study CDC25A dynamics in cultured primary cells, we also generated embryonic fibroblasts from the knock-in mice. These molecular imaging studies are in collaboration with David Piwnica-Worms and the Molecular Imaging Center at Washington University.
Translational Studies
In collaboration with investigators at Washington University School of Medicine, we performed an investigator-initiated phase 1 clinical trial monitoring the combined effects of irinotecan, a DNA-damaging agent, in combination with UCN-01, a nonspecific Chk1 inhibitor, in resistant solid-tumor malignancies. My laboratory, in collaboration with Patrick O'Connor and Edward Sausville (then at the National Cancer Institute), identified UCN-01 as a potent Chk1 inhibitor several years ago. Irinotecan inhibits topoisomerase I and induces DNA damage selectively in S-phase cells. Thus, irinotecan induces cell cycle arrest in the S and G2 phases of the cell cycle in both p53+ and p53– tumors and UCN-01 is predicted to selectively abrogate the S/G2 arrest in p53-deficient tumors. Twenty-five patients with a variety of resistant solid-tumor malignancies were enrolled in the study. All patients had failed at least two previous chemotherapeutic regimens, yet 60 percent of these patients had a clinical response (either partial response or stable disease) to the combination therapy.
The most significant effects were observed in a subset of patients with triple-negative breast cancer (TNBC: negative for estrogen receptor, progesterone receptor, and HER2 gene amplification). Of the 25 patients enrolled in the trial, 4 had TNBC and all 4 showed some response to treatment. Partial responses were observed in 2 of the 4 patients, and stable disease was observed in the other 2. This subgroup analysis provided sufficient positive evidence for the National Cancer Institute to accept a proposal to extend this phase 1 study for patients with TNBC.
TNBC is a significant clinical challenge, as ER- and HER2-targeted therapies are ineffective. The TNBC subtype has a high frequency of TP53 mutation, providing an opportunity for therapeutic intervention. Therefore, we are testing the combination of DNA-damaging agents and Chk1 inhibitors in a preclinical model of TNBC. Tumor biopsies from patients with TNBC are engrafted into the humanized mammary fat pad of immunodeficient NOD/SCID mice. TP53 is sequenced in each engrafted tumor explant, and the integrity of the p53 pathway is determined by monitoring p53 stabilization and p21 induction following DNA damage. Preliminary studies indicate that p53 status is a significant predictor of response to combination therapies involving DNA damage followed by Chk1 inhibition. Thus, tumors, including many TNBC tumors that lack a functional p53 pathway, may be effectively treated using this strategy. Collaborators on these studies include Cynthia Ma, Matthew Ellis, and Shunqiang Li (Washington University School of Medicine).
PAR-1 Protein Kinases
Establishing and maintaining cellular polarity is critical for the homeostasis of unicellular and multicellular organisms alike. The PAR (partitioning-defective) genes (PAR-1–6) were identified in Caenorhabditis elegans as essential determinants of asymmetric cell division and polarized cell growth. PAR-1 encodes a serine/threonine protein kinase, and in mammals there are four family members: PAR-1a, -1b, -1c, -1d. We are investigating the regulation and function of PAR-1 in mammals. We have observed several phenotypes in mice disrupted for PAR-1b. These include impaired fertility, growth retardation, loss of immune system homeostasis, and metabolic disorders. In contrast, mice disrupted for PAR-1a exhibit mild growth retardation but otherwise are normal. We have also demonstrated that two arms of the PKC (protein kinase C) pathway phosphorylate PAR-1 to regulate interactions with 14-3-3 proteins: one involves atypical PKC (aPKC), and the other involves novel PKCs (nPKCs) and PKD (protein kinase D). Atypical PKC phosphorylates PAR-1 on a conserved threonine residue, whereas the nPKC/PKD pathway phosphorylates PAR-1 on a conserved serine residue. Together these sites regulate 14-3-3 binding and PAR-1 localization.
Grants from the National Institutes of Health and the Susan G. Komen Foundation provided support for some of these projects.
Last updated February 12, 2010
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