The crosstalk between inflammation and tumorigenesis is now clearly established. However, how inflammation is elicited in the metastatic environment and the corresponding contribution of innate immunity pathways in suppressing tumor growth at secondary sites are poorly understood. Here, we show that mice deficient in Nlrp3 inflammasome components had exacerbated liver colorectal cancer metastatic growth, which was mediated by impaired interleukin-18 (IL-18) signaling. Control of tumor growth was independent of differential cancer cell colonization or proliferation, intestinal microbiota effects, or tumoricidal activity by the adaptive immune system. Instead, the inflammasome-IL-18 pathway impacted maturation of hepatic NK cells, surface expression of the death ligand FasL, and capacity to kill FasL-sensitive tumors. Our results define a regulatory signaling circuit within the innate immune system linking inflammasome activation to effective NK-cell-mediated tumor attack required to suppress colorectal cancer growth in the liver.

The adenoviral early region 4 open reading frame 4 (E4orf4) death factor induces p53-independent apoptosis in many cell types and appears to kill selectively transformed cells. Here we show that expression of E4orf4 in transformed epithelial cells results in early caspase-independent membrane blebbing, associated with changes in the organization of focal adhesions and actin cytoskeleton. Evidence that E4orf4 can associate with and modulate Src family kinase activity, inhibiting Src-dependent phosphorylation of focal adhesion kinase (FAK) and paxillin while increasing phosphorylation of cortactin and some other cellular proteins, is presented. Furthermore, E4orf4 dramatically inhibited the ability of FAK and c-src to cooperate in induction of tyrosine phosphorylation of cellular substrates, suggesting that E4orf4 can interfere with the formation of a signaling complex at focal adhesion sites. Consistent with a functional role for E4orf4–Src interaction, overexpression of activated c-src dramatically potentiated E4orf4-induced membrane blebbing and apoptosis, whereas kinase dead c-src constructs inhibited E4orf4 effects on cell morphology and death. Moreover treatment of E4orf4-expressing cells with PP2, a selective Src kinase inhibitor, led to inhibition of E4orf4-dependent membrane blebbing and later to a marked decrease in E4orf4-induced nuclear condensation. Taken together, these observations indicate that expression of adenovirus 2 E4orf4 can initiate caspase-independent extranuclear manifestations of apoptosis through a modulation of Src family kinases and that these are involved in signaling E4orf4-dependent apoptosis. This study also suggests that Src family kinases are likely to play a role in the cytoplasmic execution of apoptotic programs.

Cellular inhibitors of apoptosis proteins (cIAPs) are essential regulators of cell death and immunity. The corresponding contributions of IAPs to infectious disease outcomes are relatively unexplored. We find that mice deficient in cIAP2 exhibit increased susceptibility and mortality to influenza A virus infection. The lethality was not due to impaired antiviral immune functions, but rather because of death-receptor-induced programmed necrosis of airway epithelial cells that led to severe bronchiole epithelial degeneration, despite control of viral replication. Pharmacological inhibition of RIPK1 or genetic deletion of Ripk3, both kinases involved in programmed necrosis, rescued cIAP2-deficient mice from influenza-induced lethality. Genetic deletion of the death receptor agonists Fas ligand or TRAIL from the hematopoietic compartment also reversed the susceptibility of cIAP2-deficient mice. Thus, cIAP2-dependent antagonism of RIPK3-mediated programmed necrosis critically protects the host from influenza infection through maintenance of pulmonary tissue homeostasis rather than through pathogen control by the immune system.

In transformed cells, induction of apoptosis by adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) correlates with accumulation of E4orf4 in the cell membrane–cytoskeleton fraction. However, E4orf4 is largely expressed in nuclear regions before the onset of apoptosis. To determine the relative contribution of nuclear E4orf4 versus membrane-associated E4orf4 to cell death signaling, we engineered green fluorescent fusion proteins to target E4orf4 to specific cell compartments. The targeting of Ad2 E4orf4 to cell membranes through a CAAX-box or a myristylation consensus signal sufficed to mimic the fast Src-dependent apoptotic program induced by wild-type E4orf4. In marked contrast, the nuclear targeting of E4orf4 abolished the early induction of extranuclear apoptosis. However, nuclear E4orf4 still induced a delayed cell death response independent of Src-like activity and of E4orf4 tyrosine phosphorylation. The zVAD.fmk-inhibitable caspases were dispensable for execution of both cell death programs. Nevertheless, both pathways led to caspase activation in some cell types through the mitochondrial pathway. Finally, our data support a critical role for calpains upstream in the death effector pathway triggered by the Src-mediated cytoplasmic death signal. We conclude that Ad2 E4orf4 induces two distinct cell death responses, whose relative contributions to cell killing may be determined by the genetic background.

Adenovirus type 2 (Ad2) early region 4 ORF4 (E4orf4) triggers a major death pathway that requires its accumulation in cellular membranes and its tyrosine phosphorylation. This program is regulated by Src family kinases and triggers a potent ZVAD (benzyloxycarbonyl-VAD)-and Bcl2-resistant cell death response in humantransformed cells. How E4orf4 deregulates Src-dependent signaling is unknown. Here we provide strong evidence that a physical interaction requiring the kinase domain of Src and the arginine-rich motif of E4orf4 is involved. The Src binding domain of E4orf4 overlaps with, but is distinct from that of the B␣ subunit of protein phosphatase 2A (PP2A-B␣) and some E4orf4 complexes contain both PP2A and Src. Functional assays using mutant E4orf4 revealed that deregulation of Src signaling, activation of the Jun kinase pathway, and cell blebbing were all critically dependent on Src binding. In contrast, PP2A-B␣ binding per se was not required to engage the Src-dependent death pathway but was more critical for triggering a distinct death activity. Both E4orf4 death activities were manifested within a given cell population, were typified by distinct morphological features, and contributed to overall cell killing, although to different extents in various cell types. We conclude that E4orf4 binding to the Src kinase domain leads to deregulation of Src signaling and plays a crucial role in induction of the cytoplasmic death pathway. Nonetheless, both Src and PP2A enzymes are critical targets of E4orf4 that likely cooperate to trigger E4orf4-induced tumor cell killing and whose relative contributions may vary in function of the cellular background.

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The adenovirus early region 4 ORF4 protein (E4orf4) triggers a novel death program that bypasses classical apoptotic pathways in human cancer cells. Deregulation of the cell cytoskeleton is a hallmark of E4orf4 killing that relies on Src family kinases and E4orf4 phosphorylation. However, the cytoskeletal targets of E4orf4 and their role in the death process are unknown. Here, we show that E4orf4 translocates to cytoplasmic sites and triggers the assembly of a peculiar juxtanuclear actin-myosin network that drives polarized blebbing and nuclear shrinkage. We found that E4orf4 activates the myosin II motor and triggers de novo actin polymerization in the perinuclear region, promoting endosomes recruitment to the sites of actin assembly. E4orf4-induced actin dynamics requires interaction with Src family kinases and involves a spatial regulation of the Rho GTPases pathways Cdc42/N-Wasp, RhoA/Rho kinase, and Rac1, which make distinct contributions. Remarkably, activation of the Rho GTPases is required for induction of apoptotic-like cell death. Furthermore, inhibition of actin dynamics per se dramatically impairs E4orf4 killing. This work provides strong support for a causal role for endosome-associated actin dynamics in E4orf4 killing and in the regulation of cancer cell fate. INTRODUCTIONThe study of viral proteins and the cellular pathways they perturb is a powerful strategy for identifying critical functions that regulate cell growth and survival. The human adenovirus type 2 early region 4 ORF4 (Ad2 E4orf4) is a multifunctional early viral gene product that is not essential for adenovirus infection, but it seems to play several functions to facilitate viral replication (Branton and Roopchand, 2001;Ben-Israel and Kleinberger, 2002;O'Shea et al., 2005).However, when expressed alone in transformed and tumor cells, E4orf4 induces a p53-independent cell death program that shares several apoptotic hallmarks, including phagocytosis (Lavoie et al., 1998;Shtrichman and Kleinberger, 1998;Champagne et al., 2004). Nonetheless, the mechanisms involved clearly differ from those associated with apoptosis. E4orf4 killing proceeds in absence of classical caspase activation in most cancer cell types and resists to acute overexpression of Bcl-2 and caspase inhibitors (Lavoie et al., 1998;Livne et al., 2001;Robert et al., 2002). Furthermore, mitochondria depolarization is a late event and a consequence rather than a cause of cell death. Thus, E4orf4 activates a novel death pathway, which may involve calcium signaling and calpains (Robert et al., 2002). Evidence also suggests that E4orf4 killing is tumor selective, raising a great interest for the mechanisms involved (Kleinberger, 2004).Two major cellular targets are involved in the regulation of E4orf4 killing: the protein phosphatase 2A (PP2A) and the Src family of nonreceptor tyrosine kinases (Shtrichman et al., 1999;Lavoie et al., 2000;Marcellus et al., 2000). The Srcregulated death activity of E4orf4 that we call the cytoplasmic death pathway, requires a physical interaction wi...

In transformed cells, the adenovirus E4orf4 death factor works in part by inducing a Src-mediated cytoplasmic apoptotic signal leading to caspase-independent membrane blebbing and cell death. Here we show that Src-family kinases modulate E4orf4 phosphorylation on tyrosine residues. Mutation of tyrosines 26, 42, and 59 to phenylalanines inhibited Src-induced phosphorylation of E4orf4 in vivo and in vitro but had no effect on the molecular association of E4orf4 with Src. However, in contrast to wild-type E4orf4, the nonphosphorylatable E4orf4 mutant was unable to modulate Src-dependent phosphorylation and was deficient in recruiting a subset of tyrosine-phosphorylated proteins. Indeed, the Src substrates cortactin and p62dok were found to associate with wild-type E4orf4 but not with the nonphosphorylatable E4orf4. Importantly, the nonphosphorylatable mutant E4orf4 was preferentially distributed in the cell nucleus, was unable to induce membrane blebbing, and had a highly impaired killing activity. Conversely, an activated form of E4orf4 was obtained by mutation of tyrosine 42 to glutamic acid. This pseudophosphorylated mutant E4orf4 was enriched in the cytoplasm and plasma membrane, showed increased binding to phosphotyrosine-containing proteins, and induced a dramatic blebbing phenotype associated with increased cell death. Altogether, our findings strongly suggest that Srcmediated phosphorylation of adenovirus type 2 E4orf4 is critical to promoting its cytoplasmic and membrane localization and is required for the transduction of E4orf4-Src-dependent induction of membrane blebbing. We propose that E4orf4 acts in part by uncoupling Src-dependent signals to drive the formation of a signaling complex that triggers a cytoplasmic death signal.Apoptosis is a cell suicide program that plays a crucial role in the maintenance of cellular integrity (77). Two classical pathways for induction of apoptosis exist in mammalian cells, the intrinsic, or mitochondrial, and extrinsic, or death receptor, pathways, and both involve the activation of caspases, a family of cysteine proteases with aspartate specificity (3,4,28,39). This self-amplifying caspase cascade culminates in the proteolytic inactivation of critical components of survival pathways and activation of proapoptotic functions, which altogether lead to the disassembly of the cell. Despite the general role of caspases in apoptotic processes, several modes of caspaseindependent induction of cell death also exist, but the mechanisms involved are poorly understood. In general, the cytosolic hallmarks of apoptosis predominate (e.g., rounder and shrunken morphology, deformations of the plasma membrane, and membrane blebs) and are associated with DNA condensation but not with classical DNA degradation. This programmed cell death response has been termed type II apoptosis. Notably, such suicide programs can be driven by the growth suppressor PML (63), the c-myc-interacting protein Bin1 (17), the Fas-binding protein Daxx (7), and the adenoviral death factor E4orf4 (42). Caspase-...