Claudia Champagne

The Nlrp3 Inflammasome Suppresses Colorectal Cancer Metastatic Growth in the Liver by Promoting Natural Killer Cell Tumoricidal Activity

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.

Distinct cell death pathways triggered by the adenovirus early region 4 ORF 4 protein

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.

Cytoplasmic Death Signal Triggered by Src-Mediated Phosphorylation of the Adenovirus E4orf4 Protein

ABSTRACT 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 Src-mediated 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.

Regulation of Cell Death by Recycling Endosomes and Golgi Membrane Dynamics via a Pathway Involving Src-family kinases, Cdc42 and Rab11a

Actin dynamics and membrane trafficking influence cell commitment to programmed cell death through largely undefined mechanisms. To investigate how actin and recycling endosome (RE) trafficking can engage death signaling, we studied the death program induced by the adenovirus early region 4 open reading frame 4 (E4orf4) protein as a model. We found that in the early stages of E4orf4 expression, Src-family kinases (SFKs), Cdc42, and actin perturbed the organization of the endocytic recycling compartment and promoted the transport of REs to the Golgi apparatus, while inhibiting recycling of protein cargos to the plasma membrane. The resulting changes in Golgi membrane dynamics that relied on actin-regulated Rab11a membrane trafficking triggered scattering of Golgi membranes and contributed to the progression of cell death. A similar mobilization of RE traffic mediated by SFKs, Cdc42 and Rab11a also contributed to Golgi fragmentation and to cell death progression in response to staurosporine, in a caspase-independent manner. Collectively, these novel findings suggest that diversion of RE trafficking to the Golgi complex through a pathway involving SFKs, Cdc42, and Rab11a plays a general role in death signaling by mediating regulated changes in Golgi dynamics.

The Inflammatory Caspases-1 and -11 Mediate the Pathogenesis of Dermatitis in Sharpin-Deficient Mice

Chronic proliferative dermatitis in mice (cpdm) is a spontaneous multiorgan inflammatory disorder with pathological hallmarks similar to atopic dermatitis and psoriasis in humans. Cpdm mice lack expression of SHANK-associated RH domain–interacting protein, an adaptor of the linear ubiquitin assembly complex, which acts in the NF-κB pathway to promote inflammation and protect from apoptosis and necroptosis. Although skin inflammation in cpdm mice is driven by TNF- and RIPK1-induced cell death, the contribution of initiating innate immunity sensors and additional inflammatory pathways remains poorly characterized. In this article, we show that inflammasome signaling, including the expression and activation of the inflammatory caspase-1 and -11 and IL-1 family cytokines, was highly upregulated in the skin of cpdm mice prior to overt disease onset. Genetic ablation of caspase-1 and -11 from cpdm mice significantly reduced skin inflammation and delayed disease onset, whereas systemic immunological disease persisted. Loss of Nlrp3 also attenuated skin disease, albeit more variably. Strikingly, induction of apoptosis and necroptosis effectors was sharply decreased in the absence of caspase-1 and -11. These results position the inflammasome as an important initiating signal in skin disease pathogenesis and provide novel insights about inflammasome and cell death effector cross-talk in the context of inflammatory diseases.

JNK-mediated Phosphorylation of Paxillin in Adhesion Assembly and Tension-induced Cell Death by the Adenovirus Death Factor E4orf4

The adenovirus type 2 Early Region 4 ORF4 (E4orf4) protein induces a caspase-independent death program in tumor cells involving changes in actin dynamics that are functionally linked to cell killing. Because an increase in myosin II-based contractility is needed for the death of E4orf4-expressing cells, we have proposed that alteration of cytoskeletal tension is part of the signals engaging the death pathway. Yet the mechanisms involved are poorly defined. Herein, we show that the Jun N-terminal kinase JNK is activated in part through a pathway involving Src, Rho, and ROCK (Rho kinase) and contributes to dysregulate adhesion dynamics and to kill cells in response to E4orf4. JNK supports the formation of atypically robust focal adhesions, which are bound to the assembly of the peculiar actomyosin network typifying E4orf4-induced cell death and which are required for driving nuclear condensation. Remarkably, the dramatic enlargement of focal adhesions, actin remodeling, and cell death all rely on paxillin phosphorylation at Ser-178, which is induced by E4orf4 in a JNK-dependent way. Furthermore, we found that Ser-178-paxillin phosphorylation is necessary to decrease adhesion turnover and to enhance the time residency of paxillin at focal adhesions, promoting its recruitment from an internal pool. Our results indicate that perturbation of tensional homeostasis by E4orf4 involves JNK-regulated changes in paxillin adhesion dynamics that are required to engage the death pathway. Moreover, our findings support a role for JNK-mediated paxillin phosphorylation in adhesion growth and stabilization during tension signaling.

A Functional Interplay between the Small GTPase Rab11a and Mitochondria-Shaping Proteins Regulates Mitochondrial Positioning and Polarization of the Actin Cytoskeleton Downstream of Src-Family Kinases

Background: Mitochondrial dynamics are integrated within signaling systems through ill-defined mechanisms. Results: During cytoskeletal rearrangements by Src family kinases (SFK), Rab11a modulates mitochondrial dynamics that, in turn, influence actin assembly. Conclusion: Redistribution of mitochondria near actin-rich structures is mediated by SFK and Rab11a and facilitates polarization of the cytoskeleton. Significance: A new functional connection is uncovered between membrane traffic and mitochondrial dynamics during cellular remodeling. It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.

Src-family kinase signaling, actin-mediated membrane trafficking and organellar dynamics in the control of cell fate: Lessons to be learned from the adenovirus E4orf4 death factor

Evidence has accumulated that there are different modes of regulated cell death, which share overlapping signaling pathways. Cytoskeletal-dependent inter-organellar communication as a result of protein and lipid trafficking in and out of organelles has emerged as a common, key issue in the regulation of cell death modalities. The movement of proteins and lipids between cell compartments is believed to relay death signals in part through modifications of organelles dynamics. Little is known, however, regarding how trafficking is integrated within stress signaling pathways directing organelle-specific remodeling events. In this review, we discuss emerging evidence supporting a role for regulated changes in actin dynamics and intracellular membrane flow. Based on recent findings using the adenovirus E4orf4 death factor as a probing tool to tackle the mechanistic underpinnings that control alternative modes of cell death, we propose the existence of multifunctional platforms at the endosome-Golgi interface regulated by SFK-signaling. These endosomal platforms could be mobilized during cell activation processes to reorganize cellular membranes and promote inter-organelle signaling.

A critical role for cellular inhibitor of protein 2 (cIAP2) in colitis-associated colorectal cancer and intestinal homeostasis mediated by the inflammasome and survival pathways.

Cellular inhibitors of apoptosis proteins (cIAPs) are critical arbiters of cell death and key mediators of inflammation and innate immunity. cIAP2 is frequently overexpressed in colorectal cancer and in regenerating crypts of ulcerative colitis patients. However, its corresponding functions in intestinal homeostasis and underlying mechanisms in disease pathogenesis are poorly understood. We found that mice deficient in cIAP2 exhibited reduced colitis-associated colorectal cancer tumor burden but, surprisingly, enhanced susceptibility to acute and chronic colitis. The exacerbated colitis phenotype of cIAP2-deficient mice was mediated by increased cell death and impaired activation of the regenerative inflammasome-interleukin-18 (IL-18) pathway required for tissue repair following injury. Accordingly, administration of recombinant IL-18 or pharmacological inhibition of caspases or the kinase RIPK1 protected cIAP2-deficient mice from colitis and restored intestinal epithelial barrier architecture. Thus, cIAP2 orchestrates intestinal homeostasis by exerting a dual function in suppressing cell death and promoting intestinal epithelial cell proliferation and crypt regeneration.

The Interleukin (IL)-1R1 pathway is a critical negative regulator of PyMT-mediated mammary tumorigenesis and pulmonary metastasis

ABSTRACT Breast cancer is the most common cancer in women and the second leading cause of female cancer-related deaths worldwide. Inflammation is an established hallmark of tumorigenesis and an important determinant of tumor outcome and response to therapy. With advances in cancer immunotherapy, there is an urgent need to dissect the contribution of specific immune effectors in cancer development. Here, we genetically investigated the role of the Interleukin-1 (IL-1) receptor 1 (IL-1R1) pathway in breast cancer tumorigenesis and metastasis using the MMTV-PyMT mouse model. Our results indicate that IL-1R1 signaling suppresses mammary tumor cell proliferation early in tumorigenesis and curbs breast cancer outgrowth and pulmonary metastasis. We show that PyMT/Il1r1−/− mice had a higher primary tumor burden and increased mortality rate compared with IL-1R1-sufficient PyMT control mice. This phenotype was independent of the inflammatory caspases-1/-11 but driven by IL-1α, as PyMT/Il1a−/− mice phenocopied PyMT/Il1r1−/− mice. Collectively, our results suggest that IL-1α-mediated IL-1R1 signaling is tumor-suppressive in PyMT-driven breast cancer.