Cancer is a genetic disease. The fundamental defect of cancer cells is a deregulated proliferation that results from the progressive accumulation of genetic and epigenetic alterations. The regulation of cell proliferation in multicellular organisms is complex and requires the integration of a multitude of signals, both extracelluar and intracellular. The genetic and epigenetic alterations in cancer cells invariably affect the regulatory pathways that govern the proper cellular responses to this myriad of signals. Normal proliferative cells are endowed with the abilities to choose between growth and quiescence, differentiation and apoptosis. The execution of these alternative choices is influenced by physiological factors and stress to achieve a controlled and balanced proliferation. Our research is directed at elucidating signaling pathways that allow normal cells to distinguish between proliferation, differentiation and apoptosis.
Our initial approach to the study of mammalian cell growth control was to investigate the function of gene products that are mutated in cancer. We focused on the c-Abl tyrosine kinase, which is activated in the Bcr-Abl fusion protein of human chronic myelogenous leukemia; and the retinoblastoma tumor suppressor protein (RB), which is inactivated by mutations in human retinoblastoma and osteosarcoma.
Through the years, we have identified a number of functional domains in the c-Abl protein. We have shown that c-Abl tyrosine kinase, unlike most of the other tyrosine kinases, can shuttle between the nuclear and cytoplasmic compartments because it contains both nuclear localization and nuclear export signals. The cytoplasmic c-Abl is regulated by cell adhesion and is implicated in cell migration. The nuclear c-Abl is regulated by cell adhesion as well, because there is a constant cycling of c-Abl protein in and out of the nucleus. In addition, the nuclear c-Abl tyrosine kinase is inhibited by RB, but can be activated by DNA damage after RB is inactivated. The activation of nuclear c-Abl tyrosine kinase by DNA damage is dependent on the ATM kinase, which is mutated in the human disease ataxia telangiectasia. The ATM kinase is activated by double-stranded breaks, which can be induced by ionizing radiation (IR). In response to IR, ATM activates the nuclear c-Abl tyrosine kinase to phosphorylate RNA polymerase II.
Through the discovery of c-Abl/RB interactions, we also became interested in the regulation and the function of RB. We showed that RB is phosphorylated by the cyclin-dependent kinases at multiple sites. We showed that RB contains multiple, independent protein binding pockets. We showed that different phosphorylation sites differentially regulate the different protein binding pockets in RB. We generated a constitutively active RB that cannot be inactivated by phosphorylation and showed that this RB can inhibit G1/S transition as well as S-phase progression. We showed that RB is cleaved by caspases and then degraded when cells are induced to undergo apoptosis. We showed that RB directly binds to the c-Abl kinase domains and thereby blocks the activation of nuclear c-Abl tyrosine kinase by DNA damage or other signals.
Recently, we have found that c-Abl tyrosine kinase can activate the apoptosis function of p73, which is a transcription factor related to the tumor suppressor p53. We have shown that c-Abl tyrosine kinase can stabilize the p73 protein. The activation of c-Abl and p73 is observed when cells are treated with cisplatin, the chemotherapeutic agent that crosslinks DNA. We have found that cisplatin also activates the ATM kinase to activate c-Abl and p73, contributing to the killing of damaged cells. Most interestingly, we have identified the mismatch repair proteins that normally repair replication errors in DNA, to be required for cisplatin to activate ATM. The mismatch repair proteins are not required for IR to activate AT M. While cisplatin activates ATM and c-Abl to stimulate apoptosis through p73, IR activates ATM and c-Abl but does not stimulate p73 nor apoptosis. These results generate a number of interesting questions relating to the fundamental question: “how does a cell decide to kill itself after DNA damage?”. How much damage is tolerable? What triggers apoptosis? Our findings suggest that the mismatch repair proteins may play a critical role in damage assessment and in directing ATM and c-Abl to activate apoptosis.
Because RB can regulate c-Abl activity, because c-Abl can activate p73 to induce apoptosis, and because RB is cleaved and degraded when cells are induced to die, we are currently investigating the role of RB in regulating DNA damage response. We have shown that RB can become dephosphorylated (and activated) in S-phase cells following cisplatin treatment to cause an S-phase arrest. This RB-dependent S-phase arrest, combined with the G1 and G2 checkpoint responses, protect cells against apoptosis. In the absence of RB, cells are hypersensitive to cisplatin. These results suggest that RB may also play a role in directing cell fate following DNA damage. If RB can be preserved and activated, cells would choose to arrest or differentiate, but not die. If RB is degraded, the c-Abl and p73 pathway may sensitize cells to apoptosis. Experiments are underway to test these possibilities.
Current and future research will be directed at understanding how cells recognize, assess and respond to lesions in DNA and the molecular mechanisms by which a damaged cell can distinguish between continued proliferation, permanent growth arrest and suicide. Defects in these regulatory mechanisms contribute to tumor development, because mistakes made in DNA damage responses will likely cause genome instability. The functions of these pathways will also influence how normal cells and tumor cells respond to radiotherapy and chemotherapy. Hence, understanding of these regulatory mechanisms may affect how we treat cancer in the future.
Chau, B.N., H. Borges, T-T Chen, A. Masselli, I.C. Hunton, and J. Y. J. Wang (2002). Signal dependent protection from apoptosis in mice with caspase-resistant RB. Nature Cell Biol. 4: 757-765.
Puri, P.L., K. Bhakta, L. D. Wood, A. Costanzo, J. Zhu, and J. Y. J. Wang (2002). A myogenic differentiation checkpoint activated by genotoxic stress. Nature Genet. 32:585-593.
Chau, B.N. and J.Y.J. Wang (2003). Coordinated regulation of life and death by RB. Nature Rev. Cancer 3:130-138.
Wang, J.Y.J. (2004). Controlling Abl: auto-inhibition and co-inhibition? Nature Cell Biol. 6: 3-7.
Chau, B. N., T. T. Chen, Y.Y. Wan, J. DeGregori and J.Y.J. Wang (2004). Tumor necrosis factor alpha-induced apoptosis requires p73 and c-ABL activation downstream of RB degradation. Mol. Cell Biol. 24: 4438-4447.
Wang, J.Y.J. and S. K. Cho (2004). Coordination of repair, checkpoint and cell death responses to DNA damage. Advances in Protein Chemistry, 69:101-134.
Woodring, P.J., T. Hunter, and J. Y. J. Wang (2005). Mitotic phosphorylation rescues Abl from F-actin-mediated inhibition. J. Biol. Chem. 280: 10318-10335.
Borges, H., J. Bird, K. Wasson, R. D. Cardiff, N. Varki, L. Eckmann and J. Y. J. Wang (2005). Tumor promotion by caspase-resistant RB. Proc. Acad. Natl. Sci. USA, 102: 15587-15592.
Jean Y. J. Wang received her Ph.D. in Biochemistry from U.C. Berkeley and was a Jane Coffin Child postdoctoral fellow at MIT. Honors include the Searle Scholar Award, a MERIT Award from the National Cancer Institute (NCI), and the Herbert Stern Endowed Chair. Dr. Wang is Associate Director of Basic Research for the UCSD Cancer Center and an editor for the journal Molecular and Cellular Biology. She has been elected to the Board of Directors of the American Association for Cancer Research and has served on the NCI Board of Scientific Counselors.