Mechanisms of cancer cell killing by the adenovirus E4orf4 protein

Abstract

During adenovirus (Ad) replication the Ad E4orf4 protein regulates progression from the early to the late phase of infection. However, when E4orf4 is expressed alone outside the context of the virus it induces a non-canonical mode of programmed cell death, which feeds into known cell death pathways such as apoptosis or necrosis, depending on the cell line tested. E4orf4-induced cell death has many interesting and unique features including a higher susceptibility of cancer cells to E4orf4-induced cell killing compared with normal cells, caspase-independence, a high degree of evolutionary conservation of the signaling pathways, a link to perturbations of the cell cycle, and involvement of two distinct cell death programs, in the nucleus and in the cytoplasm. Several E4orf4-interacting proteins including its major partners, protein phosphatase 2A (PP2A) and Src family kinases, contribute to induction of cell death. The various features of E4orf4-induced cell killing as well as studies to decipher the underlying mechanisms are described here. Many explanations for the cancer specificity of E4orf4-induced cell death have been proposed, but a full understanding of the reasons for the different susceptibility of cancer and normal cells to killing by E4orf4 will require a more detailed analysis of the complex E4orf4 signaling network. An improved understanding of the mechanisms involved in this unique mode of programmed cell death may aid in design of novel E4orf4-based cancer therapeutics.

Figure 1

Mutation analysis of PP2A and Src binding sites in E4orf4. The Ad5 E4orf4 protein sequence is shown. The basic E4ARM domain is displayed in bold and underlined in red. The tyrosines that are phosphorylated by Src kinases are in bold green and a larger font. Other residues involved in E4orf4-induced cell death are marked in pink. Mutations in the E4orf4 sequence that reduced association of E4orf4 with a PP2A phosphatase activity by at least two-fold and impaired E4orf4-induced cell death (‘class I’ mutants [11]) are shown above the sequence in light blue. Mutations that did not reduce the E4orf4-PP2A interaction more than two-fold but were deficient in induction of cell death (‘class II mutants’) are shown below the sequence in dark red. Three more mutations were found to reduce PP2A binding: V19A/T102I, A25T/ΔD52/R87C and ΔV29/R81C, which are not shown for simplicity sake. The basic E4orf4 ARM domain is required for Src kinase binding, but has only a low effect on PP2A binding [14,19].

Figure 2

E4orf4-induced cell death is highly conserved in evolution. E4orf4-induced toxicity was studied in the yeast S. cerevisiae, in Drosophila melanogaster and in mammalian cells in tissue culture. The pictures shown here demonstrate E4orf4 effects in the various organisms, including budding defects in yeast (marked with arrows), reduced size of yeast colonies, caspase activation in a Drosophila wing disc, a wing defect in adult flies (marked with an arrow) and nuclear condensation seen by DAPI staining with cell rounding and blebbing seen by GFP staining in mammalian cells (arrows mark nuclei of transfected cells). E4orf4-induced toxicity required an interaction with PP2A in all these organisms and an interaction with Src kinases contributed to this process in flies and mammals. Studies of E4orf4 in all these model systems contributed different insights into E4orf4 biology. See the text for more details. Parts of this figure were adapted from the following sources with permissions: Top panel: [45]. Middle panel: [22]. Bottom panel: [25].

Figure 3

A working model of E4orf4 function in chromatin. Various SNF2h-containing complexes participate in chromatin remodeling and affect transcription, DNA replication, DNA repair, etc. The model shown in this figure proposes that E4orf4 together with PP2A inhibits the ACF chromatin remodeler. This inhibition leads to a shift in the balance between various SNF2h-containing remodeling complexes, allowing, for example, more activity of a WSTF-SNF2h complex. The variation in chromatin remodeling activity alters chromatin structure and induces changes in transcription, DNA replication, DNA repair, or other processes that require remodeling. These events contribute to E4orf4 functions during virus infection and lead to cell death when E4orf4 is expressed alone. This figure is adapted from [57] with permission of Oxford University Press.

Figure 4

An integrated model of the mechanisms underlying E4orf4-induced cell death. E4orf4, in collaboration with its partners, PP2A and Src, induces alterations in the nucleus (chromatin remodeling, perturbations in cell cycle regulation) and in the cytoplasm (actin remodeling, changes in protein and membrane trafficking, changes in mitochondria morphology and mobilization), which result in blebbing, nuclear condensation and cell death: see the text for details. Arrows representing PP2A-dependent signaling are marked in orange and arrows showing Src-dependent signaling are marked in blue. Nuclear effectors are highlighted in orange and non-nuclear effectors are highlighted in blue. Connections in the E4orf4 network that were suggested but not proven yet are shown by discontinuous arrows. E4orf4 is represented by a structural model containing three alpha-helices as suggested by ab initio modeling [12]. This model was originally published in J. Biol. Chem. by Ben Horowitz, Rakefet Sharf, Meirav Avital-Shacham, Antonina Pechkovsky, and Tamar Kleinberger. 2013. 288: 13718-13727. © The American Society for Biochemistry and Molecular Biology.