|Ph.D Student||Lahav Maoz|
|Subject||Identification and Characterization of the Signal|
Transduction Pathway Induced by the Viral Protein
E4orf4 in Drosophila
|Department||Department of Medicine||Supervisor||Professor Tamar Kleinberger|
|Full Thesis text|
E4orf4 is a 14kDa protein encoded by adenovirus. Within the context of the virus, E4orf4 contributes to temporal regulation of the progression of viral infection through processes which include down regulation of signal transduction pathways induced by adenovirus, control of gene expression and alternative splicing of adenovirus mRNA. When expressed individually, E4orf4 induces apoptosis in transformed cells. Cell death induced by E4orf4 is unique by at least three criteria. First, oncogenic transformation of primary cells in tissue culture sensitizes them to cell killing by E4orf4, indicating that E4orf4 research may have exciting implications for cancer therapy. Second, E4orf4-induced cell death does not require the presence of the p53 tumor suppressor gene. Mutations in p53 or in cellular proteins that modulate its expression or activity are found in most human cancers, indicating that loss of p53 activity is crucial for human tumorigenesis. In many cases, the inability of p53- deficient cancer cells to respond to DNA damage by induction of apoptosis leads to their resistance to chemotherapy. Therefore, activation of a p53-independent apoptotic pathway would be beneficial. A third unique feature of E4orf4-induced apoptosis is that it is caspase-independent in many cell lines. Thus E4orf4-induced apoptosis is a non-classical type of apoptosis. Instead of a dependence on caspases, E4orf4-induced apoptosis requires an interaction of the viral protein with the cellular protein phosphatase PP2A and with kinases of the Src family and these interactions promote cell death. However, the physiologically relevant downstream targets of E4orf4-PP2A have not been identified to date while the E4orf4-Src downstream targets are proposed to be activated in part through the Jun N-terminal kinase (JNK) pathway. Yet the mechanisms involved in cell death are poorly defined. The three unique features of E4orf4-induced apoptosis suggest that the E4orf4-initiated pathway includes components that respond differentially to the E4orf4 signal in cancer and normal cells, and are not part of the widely studied classical apoptotic pathways. Exploiting components of this pathway as cancer drug targets could address situations in which the currently used anti-cancer drugs are not efficient. Thus, identification and study of E4orf4 network components in cancer and normal cells is important and may contribute to the identification of novel drug targets for cancer therapy.
Many viral proteins act on conserved components of signaling pathways. One consequence of this conservation is that genetic model organisms such as Drosophila melanogaster can serve for analyzing and investigating the mechanisms underlying functions of viral proteins. In this study, we employ the powerful genetic tools provided by the Drosophila model organism to characterize the activities of E4orf4 in normal and cancer tissues in vivo. We show that E4orf4 induces a low level of apoptosis in normal tissues in the fly, generating aberrant morphologies, such as small, rough eyes (GMR>E4orf4) or wings lacking their posterior parts (en>E4orf4). E4orf4-induced cell death results from both caspase-dependent and caspase- independent apoptosis. In addition, E4orf4-expressing normal tissues are induced to undergo compensatory proliferation, which may reduce E4orf4 tissue damage. We further show that the interactions of E4orf4 with the PP2A-B55 subunit and with Src kinases are conserved in evolution and are required additively for E4orf4-induced apoptosis in the fly, similarly to the situation in mammalian cells.
Most importantly, using mosaic animals with GFP-marked eye clones containing mutations in tumor suppresors and/or overexpressing oncogenic RasV12, we show that E4orf4 can kill cells of various types of tumors including aggressive, invasive tumors much more efficiently than normal WT cells.
Given the conservation of the mechanisms examined here and the selective nature of E4orf4 toward malignant cells in a multicellular organism, E4orf4 may prove to be an anti-cancer agent in higher organisms including ourselves.