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Andrew Thorburn, Professor and Chair

Ph.D. (1990) University of Oxford



Department of Pharmacology
Molecular Biology
Campus Box 8303
RC1 South, L18-6100
Phone: 303-724-3663

Andrew.Thorburn@ucdenver.edu

Our laboratory studies the regulation of apoptosis and other forms of cell death as it relates to two important issues in cancer biology¬– the development of cancer and the response of cancer cells to therapy. Many of the projects in our lab focus on understanding proteins that contain Death Domains (DD), which are protein interaction domains that are particularly important in the regulation of apoptosis by Death Receptors of the Tumor Necrosis Factor superfamily. Related projects address the following questions.

Which apoptosis signaling pathways must be disrupted to allow epithelial cancers to develop? Evasion of apoptosis is one of the hallmarks of cancer and defects in apoptosis regulation are thought to be necessary for cancer development. Thus, it is sometimes thought that cancer cells are generally resistant to apoptotic stimuli in comparison with their normal counterparts. However, this idea is incorrect; most cancer cells undergo apoptosis quite readily and are, in fact, often more sensitive than their normal counterparts to common apoptotic stimuli such as DNA damaging agents and other stresses. This raises the question of which, if any, specific apoptotic pathways must be disrupted to permit cancer development. We tackled this problem by searching for signaling proteins that induce apoptosis in primary cultures of normal human epithelial cells (from prostate or breast) but cannot induce apoptosis in cancer cells. The molecule that we found is the DD from an adaptor protein called FADD, which is an essential component of death receptor signaling pathways. The isolated FADD-DD can induce apoptosis in normal but not cancerous epithelial cells and does not kill normal fibroblasts or other cell types. As far as we are aware, this is the first example of an apoptosis pathway that is epithelial-specific, functional in normal primary cells and disabled in cancer cells. These characteristics are what we expect for a cell death pathway that functions to suppress cancer and is specifically disrupted during carcinoma development. The FADD-DD-dependent pathway in normal cells is mechanistically distinct from the established FADD pathway, which works in cancer cells after stimulation with Death Receptor agonists. The novel pathway involves both caspases and serine proteases that kill cells by different mechanisms involving both apoptosis and an alternate form of programmed cell death called autophagy. The pathway becomes defective at a very early stage in the transformation process (acquisition of an immortalized phenotype) and resistance to the pathway is not related to loss of p53 function activation of telomerase, inactivation of the Rb gene or loss of function of the INK4a/ARF locus. Therefore, we have identified a novel p53-independent, epithelial-specific, cell death pathway that becomes defective upon epithelial cell immortalization and is perhaps the earliest cell death defect during breast or prostate cancer development. Current work is focused on identifying components of this signaling pathway, determining the role of death receptor agonists in its activation and determining why immortal cells are resistant to the pathway. We are also developing mouse models to test how this pathway is involved in tumor suppression in vivo.

How do Fas and TRAIL receptors work in normal and cancerous cells? We are studying the mechanism of death receptor activation by focusing on the recruitment of FADD to activated death receptors. Using modified two hybrid methods that allow us to identify point mutants that specifically disrupt some protein-protein interactions, we have shown that current models of how FADD is recruited to these receptors through Death Domains on both molecules are oversimplified and that regulation through other domains in the receptors and FADD is an important aspect of receptor regulation. In collaboration with Human Genome Sciences, which has developed therapeutic antibodies that activate TRAIL receptors and are in phase I trials for various human cancers, we are studying how these domains regulate receptor signaling pathways in normal and cancer cells. Current work focuses on understanding the mechanisms through which the other domains control DD interactions, determining the role of the receptors in activating the novel FADD-DD pathway in normal cells and identifying and understanding tumor-derived mutations in receptors and FADD that affect these signaling mechanisms.

How and why does the adaptor protein TRADD induce apoptotic signals from different cellular compartments? Another DD-containing adaptor protein, TRADD, is an essential component of the TNFR1 signaling pathway. Our work on TRADD focuses on understanding how TRADD signaling is regulated in different sub-cellular compartments. We made the surprising discovery that TRADD, a protein that had been expected to work at the cell surface TNFR1 receptor, is rapidly shuttled between the cytoplasm and the nucleus. Furthermore, when in the nucleus, TRADD associates with PML nuclear bodies and can induce apoptosis through a mechanism that is different from its mode of action in the cytoplasm. This is therefore an unusual example where the same protein activates different cell death pathways depending upon its location in the cell. The nuclear apoptosis pathway that is activated by TRADD requires PML and involves p53 suggesting that this pathway is involved in nuclear stress responses. Current work focuses on understanding the mechanism of action of the nuclear TRADD pathway, and we have shown that this occurs via activation of the mitochondrial apoptosis pathway leading to activation of caspase-9. We are also determining how and why TRADD nuclear shuttling is regulated and determining the physiological role of this pathway.

How do tumor cell-targeted toxins induce apoptosis? In a translational research project in collaboration with Dr. Art Frankel, we are studying the mechanism of action of tumor-cell targeted toxins that are being developed to treat glioblastoma and various leukemias. These molecules contain a targeting domain (usually a growth factor or antibody) fused to a bacterial or plant toxin (in our case diphtheria toxin). Upon receptor-mediated uptake into tumor cells, the toxin induces apoptosis. We are studying how this occurs with a particular focus on the mechanism of action of an EGF-targeted diphtheria toxin that we are developing as a treatment for glioblastoma. In some cells FADD-dependent signaling pathways are important for toxin-induced cell killing and caspase-dependent apoptosis occurs. However in glioblastoma cells, one of our toxins is an effective cell killer but does so through a caspase-independent mechanism. Moreover, the targeted diphtheria toxin can selectively sensitize glioblastoma cells to TRAIL and agonistic antibodies that activate the TRAIL receptors. Thus, we have a system where we can alter both the extent and mechanism of glioblastoma cell killing by treating cells with the recombinant toxin on its own or in combination with TRAIL receptor agonists. Current work focuses on understanding how these toxins work, determining the mechanisms of caspase-independent cell killing and synergy with TRAIL. Future studies will focus on determining how these mechanisms impact on the clinical response to the drug.

Bharadwaj, S., Hitchcock-DiGregori, S., Thorburn, A. and Prasad, G.L. (2004). “Amino terminus is essential for Tropomyosin functions: N-terminal modification disrupts stress fiber organization and abolishes anti-oncogenic effects of Tropomyosin-1.” J. Biol. Chem. 279, 14039-14048.

Thomas, L.R., Henson, A. Reed, J.C., Salsbury, F. R., and Thorburn, A. (2004). “Direct binding of FADD to the TRAIL receptor DR5 is regulated by the death effector domain of FADD.”J. Biol. Chem. In Press. doi: 10.1074/jbc.M401680200

Bender, L.M., Morgan, M.J., Thomas, L.R. Liu, Z-G., and Thorburn, A. (2005) “The adaptor protein TRADD activates distinct mechanisms of apoptosis from the nucleus and cytoplasm.” Cell Death & Differentiation. 12, 473-481.

Thorburn, J., Moore, F., Rao, A, Barclay, W., Thomas, L.R., Grant, K.W, Cramer, S.D. and Thorburn, A. (2005) “Selective inactivation of a FADD-dependent apoptosis and autophagy pathway in immortal epithelial cells.” Mol. Biol. Of the Cell.  16, 1189-1199.

Bender, L.M. and Thorburn, A. (2005) “Death Domain interactions in Death Receptor Signaling” (2005) Journal of Biological Sciences. 5, 33-37.

Iordanov MS, Kirsch JD, Ryabinina OP, Wong J, Spitz PN, Korcheva VB, Thorburn A, Magun BE. (2005)  “Recruitment of TRADD, FADD, and caspase 8 to double-stranded RNA-triggered death inducing signaling complexes (dsRNA-DISCs)”. Apoptosis  10, 167-176.

Liu, T.-F., Hall, P.D., Cohen, K.A., Willingham, M.C., Cai, J., Thorburn, A., and Frankel, A.E. (2005). “Interstitial diphtheria toxin-epidermal growth factor fusion protein therapy produces regressions of human glioblastoma multiforme tumors in athymic nude mice.”  Clinical Cancer Research. 11, 329-334.

Thorburn, A. (2005) “Apoptosis induction by tumor targeted toxins.”  In Application of Apoptosis to Cancer Treatment, ed. M. Sluyser. Springer Science and Business media. 
 
Alappat, E.C., Feig. C., Volkland, J., Boterinas, B., Samuels, M., Murmann, A.E., Thorburn, A., Kidd, V.J., Slaughter, C.A., Osborne, S.L., Winoto, A., Tang, W., Peter, M.E., (2005). “Phosphorylation of FADD at Serine 194 by CKIa regulates its nonapoptotic activities.” Molecular Cell 19, 321-232.

Thomas, L.R., Bender, L.M., Morgan, M.J., and Thorburn, A.  (2006) “Extensive regions of the FADD death domain are required for binding to the TRAIL receptor DR5.”  Cell Death & Differentiation 13, 160-162.

Thorburn, A. and Frankel, A.E. (2006) “Apoptosis and anthracycline cardiotoxicity” Molecular Cancer Therapeutics. 5, 197-199.

Zhao, W., Kridel, S., Thorburn, A., Kooshli, M., Little, J., Hebbar, S., and Robbins, M. (2006) Fatty acid synthase: a novel target for antiglioma therapy. Br. Journal of Cancer 95, 869-878.

Hippert, M.M., O’Toole, P.S. and Thorburn, A. (2006) “Autophagy in cancer: good bad or both?”  Cancer Research 66, 9349-9351.

Behbakht, K., Qamar, L., Aldridge, C., Coletta, R.O., Davidson, S.A., Thorburn, A., and Ford, H.L. (2007)  “Six1 over-expression in ovarian carcinoma causes resistance to TRAIL-mediated apoptosis and is associated with poor survival.”  Cancer Research  67, 3036-3042.

Thorburn, A. (2007) “Tumor Necrosis factor-related Apoptosis Inducing Ligand (TRAIL) Pathway Signaling.” Journal of Thoracic Oncology 2, 461-465.

Lyone, T.R., Thorburn, J., Ryan, P.W. Thorburn, A., Anderson, S.M. and Kassenbrock, C.K. (2007) “Regulation of the pro-apoptotic scaffolding protein POSH by Akt.”  Journal of Biological Chemistry, 282, 21987-21997.

Bin, L., Thorburn, J, Thomas, L.R. Clark, P.E., Humphreys R., and Thorburn, A. (2007)  “Tumor-derived mutations in the TRAIL receptor DR5 inhibit TRAIL signaling through the DR4 receptor by competing for ligand binding.” Journal of Biological Chemistry, In Press.

Horita, H., Frankel, A.E. and Thorburn, A. (2007) “Acute Myeloid Leukemia-targeted toxins kill tumor cells by cell type-specific mechanisms and synergize with TRAIL to allow manipulation of the extent and mechanism of tumor cell death.  Leukemia.  In Press.


Latest Publications in PubMed