Mechanisms of Cancer Cell Killing by the Adenovirus  E4orf4 Protein

Kleinberger, Tamar

doi:10.3390/v7052334

Cited by 13 publications

(17 citation statements)

References 92 publications

(252 reference statements)

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“…It has been reported that when E4orf4 is expressed alone, it induces a unique mode of cancer-specific cell death [ 60 , 61 ]. Inhibition of the DDR may contribute to this process.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, E4orf4-induced cell death is more efficient in oncogene-transformed cells in tissue culture than in normal cells [ 60 ], indicating that study of E4orf4 signaling may have implications for cancer therapy. The basis for the cancer specificity of E4orf4-induced cell death will be better understood once knowledge of the underlying mechanisms is improved [ 61 ].…”

Section: Introductionmentioning

confidence: 99%

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The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response

et al. 2016

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The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition by E4orf4 contributes both to the efficiency of adenovirus replication and to the ability of E4orf4 to kill cancer cells.

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“…It has been reported that when E4orf4 is expressed alone, it induces a unique mode of cancer-specific cell death [ 60 , 61 ]. Inhibition of the DDR may contribute to this process.…”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response

et al. 2016

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“…Protein phosphatase 2A (PP2A) is an important protein partner of E4orf4 which contributes to several E4orf4 functions, including attenuation of the DDR (26,39,(49)(50)(51)(52)(53). PP2A is a heterotrimeric enzyme containing a catalytic subunit (C), a scaffolding subunit (A), and one of several regulatory B subunits that belong to at least four unrelated gene families and which determine the specificity of the PP2A holoenzyme toward various substrates (54).…”

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confidence: 99%

Biphasic Functional Interaction between the Adenovirus E4orf4 Protein and DNA-PK

Nebenzahl-Sharon

Shalata

Sharf

et al. 2019

J Virol

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The adenovirus (Ad) E4orf4 protein contributes to virus-induced inhibition of the DNA damage response (DDR) by reducing ATM and ATR signaling. Consequently, E4orf4 inhibits DNA repair and sensitizes transformed cells to killing by DNA-damaging drugs. Inhibition of ATM and ATR signaling contributes to the efficiency of virus replication and may provide one explanation for the cancer selectivity of cell death induced by the expression of E4orf4 alone. In this report, we investigate a direct interaction of E4orf4 with the DDR. We show that E4orf4 physically associates with the DNA-dependent protein kinase (DNA-PK), and we demonstrate a biphasic functional interaction between these proteins, wherein DNA-PK is required for ATM and ATR inhibition by E4orf4 earlier during infection but is inhibited by E4orf4 as infection progresses. This biphasic process is accompanied by initial augmentation and a later inhibition of DNA-PK autophosphorylation as well as by colocalization of DNA-PK with early Ad replication centers and distancing of DNA-PK from late replication centers. Moreover, inhibition of DNA-PK improves Ad replication more effectively when a DNA-PK inhibitor is added later rather than earlier during infection. When expressed alone, E4orf4 is recruited to DNA damage sites in a DNA-PK-dependent manner. DNA-PK inhibition reduces the ability of E4orf4 to induce cancer cell death, likely because E4orf4 is prevented from arriving at the damage sites and from inhibiting the DDR. Our results support an important role for the E4orf4 -DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.IMPORTANCE Several DNA viruses evolved mechanisms to inhibit the cellular DNA damage response (DDR), which acts as an antiviral defense system. We present a novel mechanism by which the adenovirus (Ad) E4orf4 protein inhibits the DDR. E4orf4 interacts with the DNA damage sensor DNA-PK in a biphasic manner. Early during infection, E4orf4 requires DNA-PK activity to inhibit various branches of the DDR, whereas it later inhibits DNA-PK itself. Furthermore, although both E4orf4 and DNA-PK are recruited to virus replication centers (RCs), DNA-PK is later distanced from late-phase RCs. Delayed DNA-PK inhibition greatly contributes to Ad replication efficiency. When E4orf4 is expressed alone, it is recruited to DNA damage sites. Inhibition of DNA-PK prevents both recruitment and the previously reported ability of E4orf4 to kill cancer cells. Our results support an important role for the E4orf4 -DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.

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“…However, there are no publications to suggest that ADP-mediated lysis might represent a necrotic process nor that ADP interacts with RIPK3 or MLKL. E4orf4, when expressed alone, can induce p53-independent, caspase-independent cell death 31 , 32 , but again, there is no evidence to support an interaction with any necrotic proteins, nor indeed to support a role for E4orf4 in cell death during productive adenovirus infection 33 .…”

Section: Discussionmentioning

confidence: 97%

RIPK3 promotes adenovirus type 5 activity

et al. 2017

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Oncolytic adenoviral mutants infect human malignant cells and replicate selectively within them. This induces direct cytotoxicity that can also trigger profound innate and adaptive immune responses. However, the mechanism by which adenoviruses produce cell death remains uncertain. We previously suggested that type 5 adenoviruses, including the E1A CR2 deletion mutant dl922-947, might induce a novel form of programmed death resembling necroptosis. Here we have investigated the roles of core necrosis proteins RIPK1, RIPK3 and MLKL in the cytotoxicity of dl922-947 and other adenovirus serotypes. By electron microscopy, we show that dl922-947 induces similar necrotic morphology as TSZ treatment (TNF-α, Smac mimetic, zVAD.fmk). However, dl922-947-mediated death is independent of TNF-α signalling, does not require RIPK1 and does not rely upon the presence of MLKL. However, inhibition of caspases, specifically caspase-8, induces necroptosis that is RIPK3 dependent and significantly enhances dl922-947 cytotoxicity. Moreover, using CRISPR/Cas9 gene editing, we demonstrate that the increase in cytotoxicity seen upon caspase inhibition is also MLKL dependent. Even in the absence of caspase inhibition, RIPK3 expression promotes dl922-947 and wild-type adenovirus type 5 efficacy both in vitro and in vivo. Together, these results suggest that adenovirus induces a form of programmed necrosis that differs from classical TSZ necroptosis.

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Mechanisms of Cancer Cell Killing by the Adenovirus E4orf4 Protein

Cited by 13 publications

References 92 publications

The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response

The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response

Biphasic Functional Interaction between the Adenovirus E4orf4 Protein and DNA-PK

RIPK3 promotes adenovirus type 5 activity

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