Abstract Caspase-4 (CASP4) is a member of the inflammatory caspase subfamily and promotes inflammation. Here, we report that CASP4 in lung adenocarcinoma cells contributes to both tumor progression via angiogenesis and tumor hyperkinesis and tumor cell killing in response to high interferon (IFN)-γ levels. We observe that elevated CASP4 expression in the primary tumor is associated with cancer progression in patients with lung adenocarcinoma. Further, CASP4 knockout attenuates tumor angiogenesis and metastasis in subcutaneous tumor mouse models. CASP4 enhances the expression of genes associated with angiogenesis and cell migration in lung adenocarcinoma cell lines through nuclear factor kappa-light chain-enhancer of activated B cell signaling without stimulation by lipopolysaccharide or tumor necrosis factor. CASP4 is induced by endoplasmic reticulum stress or IFN-γ via signal transducer and activator of transcription 1. Most notably, lung adenocarcinoma cells with high CASP4 expression are more prone to IFN-γ-induced pyroptosis than those with low CASP4 expression. Our findings indicate that the CASP4 level in primary lung adenocarcinoma can predict metastasis and responsiveness to high-dose IFN-γ therapy due to cancer cell pyroptosis.

Caspase-4 (CASP4) is a member of the inflammatory caspase subfamily and promotes inflammation. Here, we report that CASP4 in non-small cell lung cancer cells contributes to both tumor progression via angiogenesis and tumor hyperkinesis and tumor cell killing in response to high interferon (IFN)-γ levels. We revealed that elevated CASP4 expression within the primary tumor was associated with cancer progression in patients with non-small cell lung cancer. Further, CASP4 knockout attenuated tumor angiogenesis and metastasis in syngeneic mouse models. CASP4 enhanced the expression of genes associated with angiogenesis and cell migration through nuclear factor kappa-light chain-enhancer of activated B cell signaling without lipopolysaccharide or tumor necrosis factor in lung cancer cell lines. CASP4 was induced by endoplasmic reticulum stress or IFN-γ via signal transducer and activator of transcription 1. Most notably, lung cancer cells with high CASP4 expression were more prone to IFN-γ-induced cell death than those with low CASP4 expression. Our findings indicate that the CASP4 level in primary non-small cell lung cancer can predict metastasis and responsiveness to high-level IFN-γ therapy.

In addition to its role in pyroptosis and inflammatory cytokine maturation, caspase-4 (CASP4) also contributes to the fusion of phagosomes with lysosomes and cell migration. However, its role in cell division remains elusive. In this study, we demonstrate that CASP4 is indispensable for proper cell division in epithelial cells. Knockout of CASP4 (CASP4 KO) in HepG2 cells led to delayed cell proliferation, increased cell size, and increased multinucleation. In mitosis, CASP4 KO cells showed multipolar spindles, asymmetric spindle positioning, and chromosome segregation errors, ultimately increasing DNA content and chromosome number. We also found that phalloidin, a marker of filamentous actin, increased in CASP4 KO cells owing to suppressed actin depolymerization. Moreover, the levels of actin polymerization-related proteins, including Rho-associated protein kinase1 (ROCK1), LIM kinase1 (LIMK1), and phosphorylated cofilin, significantly increased in CASP4 KO cells. These results suggest that CASP4 contributes to proper cell division through actin depolymerization.

Inflammation is observed in many tumors, which affects metastasis, infiltration, and immune escape and causes poor differentiation of the cancer cells. However, the molecular basis underlying the relationship between inflammation and poor differentiation in tumors has not been identified. In this study, we demonstrate that angiopoietin-like protein-8 (ANGPTL8), which is induced by stress stimuli such as inflammation, is involved in the maintenance of the undifferentiated state of clear cell renal cell carcinoma (ccRCC) cells. ANGPTL8 is also involved in the production of chemokines that attract immune suppressor cells to the tumor microenvironment. ANGPTL8 sustains the continuous production of chemokines by activating the NF-κB signaling pathway and maintains the undifferentiated state of ccRCC cells. Finally, ANGPTL8 is induced by STAT3 signaling, which is activated by immune cells in the tumor microenvironment. These results support a role for ANGPTL8 in determining the properties of ccRCC by hampering tumor cell differentiation and establishing the tumor microenvironment.

Wnt/β-catenin is believed to regulate different sets of genes with different coactivators, cAMP response element-binding protein (CREB)-binding protein (CBP) or p300. However, the factors that determine which coactivators act on a particular promoter remain elusive. ICG-001 is a specific inhibitor for β-catenin/CBP but not for β-catenin/p300. By taking advantage of the action of ICG-001, we sought to investigate regulatory mechanisms underlying β-catenin coactivator usage in human pancreatic carcinoma PANC-1 cells through combinatorial analysis of chromatin immunoprecipitation-sequencing and RNA-sequencing. CBP and p300 preferentially bound to regions with the TCF motif alone and with both the TCF and AP-1 motifs, respectively. ICG-001 increased β-catenin binding to regions with both the TCF and AP-1 motifs, flanking the genes induced by ICG-001, concomitant with the increments of the p300 and AP-1 component c-JUN binding. Taken together, AP-1 possibly coordinates β-catenin coactivator usage in PANC-1 cells. These results would further our understanding of the canonical Wnt/β-catenin signaling divergence.

Peritoneal metastasis is a common mode of spread of ovarian cancer. Despite therapeutic advances, some patients have intractable peritoneal metastasis. Therefore, in-depth characterization of the molecular mechanism of peritoneal metastasis is a key imperative. Angiopoietin-like protein 2 (ANGPTL2) is an inflammatory factor which activates NF-κB signaling and plays an important role in the pathogenesis of various inflammatory diseases including cancers, such as lung and breast cancer. In this study, we examined the role of ANGPTL2 in ovarian cancer peritoneal metastasis. We observed no difference of cell proliferation between ANGPTL2-expressing and control cells. In the mouse intraperitoneal xenograft model, formation of peritoneal metastasis by ANGPTL2-expressing cells was significantly decreased compared to control. In the in vitro analysis, the expressions of integrin α5β1, α6, and β4, but not those of αvβ3, α3, α4, and β1, were significantly decreased in ANGPTL2-expressing cells compared to control cells. ANGPTL2-expressing cells showed significantly inhibited adherence to laminin compared to control. In addition, we observed upregulation of anoikis (a form of programmed cell death occurring under an anchorage-independent condition) and significant decrease in the expression of Bcl-2 in ANGPTL2-expressing cells as compared to control cells. These results suggest that ANGPTL2 expression in ovarian cancer cells represses peritoneal metastasis by suppressing anoikis resistance.

PD-L1 is one of the important immune checkpoint molecules that can be targeted by cancer immunotherapies. PD-L1 has a soluble form (sPD-L1) and a membrane-bound form (mPD-L1). Conventional enzyme-linked immunosorbent assay (ELISA) systems can detect sPD-L1 using anti-PD-L1 capture antibody through the antigen-antibody reaction, but cannot evaluate the quality and function of sPD-L1. In this study, we developed a novel ELISA system for the detection and quantification of sPD-L1 with PD-1-binding capacity (bsPD-L1). To capture bsPD-L1 through the ligand-receptor reaction, the anti-PD-L1 capture antibody in the conventional ELISA was replaced with PD-1-Ig fusion protein in the new ELISA. The new ELISA could detect bsPD-L1 in 29 out of 75 plasma samples from patients with non-small cell lung cancer (NSCLC), with higher sensitivity and frequency than the conventional ELISA. The western blot analysis showed that sPD-L1 in the plasma was glycosylated. Treatment of the samples with glycosidase reduced the absorbance determined by the new ELISA but had no effect on the absorbance determined by the conventional ELISA. These results suggest that glycosylation of sPD-L1 is important for its binding to the immobilized PD-1 in the new ELISA. Our new ELISA system may be useful for the evaluation of functional sPD-L1 with PD-1-binding capacity in cancer patients.

Natural infection with measles virus (MV) establishes lifelong immunity. Persistent infection with MV is likely involved in this phenomenon, as non-replicating protein antigens never induce such long-term immunity. Although MV establishes stable persistent infection in vitro and possibly in vivo, the mechanism by which this occurs is largely unknown. Here, we demonstrate that MV changes the infection mode from lytic to non-lytic and evades the innate immune response to establish persistent infection without viral genome mutation. We found that, in the persistent phase, the viral RNA level declined with the termination of interferon production and cell death. Our analysis of viral protein dynamics shows that during the establishment of persistent infection, the nucleoprotein level was sustained while the phosphoprotein and large protein levels declined. The ectopic expression of nucleoprotein suppressed viral replication, indicating that viral replication is self-regulated by nucleoprotein accumulation during persistent infection. The persistently infected cells were able to produce interferon in response to poly I:C stimulation, suggesting that MV does not interfere with host interferon responses in persistent infection. Our results may provide mechanistic insight into the persistent infection of this cytopathic RNA virus that induces lifelong immunity.

Sepsis is a systemic inflammatory response to infection associated with multiple organ dysfunction syndrome and a high mortality rate. In septic shock induced by severe peritonitis, early response of peritoneal macrophages against infected microbes is vital in preventing the spread of infection. We found that the mucosal homing receptor CCR9, is induced in peritoneal macrophages in response to inflammatory stimulation. We used a cecal ligation and puncture (CLP) model of sepsis to determine the role of CCR9 with respect to peritoneal macrophages, and controlling peritoneal infection and systemic inflammation. CCR9−/− mice showed aggravated septic shock with higher mortality rates compared with wild-type (WT) mice. Six hours after CLP, CCR9−/− mice demonstrated a greater inflammatory response. This was associated with higher production of inflammatory cytokines, such as IL-6, TNF and IP-10 in peritoneal lavage compared with WT mice. Although the numbers of peritoneal bacteria were elevated in CCR9−/− mice subjected to CLP compared with WT mice, this was normalized in CCR9−/− mice subjected to CLP through the adoptive transfer of WT peritoneal macrophages. We conclude that CCR9 is required for recruitment of peritoneal macrophages in the steady state to control systemic sepsis during early phases of peritoneal infection.

Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites.

To maintain immune homeostasis in the intestine, the intestinal immune system has evolved several tolerogenic mechanisms toward intestinal microflora and food antigens. Although programmed cell death-1 (PD-1) protein has been implicated in immunological tolerance in the intestine and gut-associated lymphoid tissues (GALTs), distribution of its ligands PD-L1 and PD-L2 in the small intestine lamina propria (LP) are unknown. We investigated PD-L1 expression in intestinal LP and found that IgA plasma cells (PCs) were major PD-L1 expressing cells. PD-L1 expression levels on IgA PCs were higher than that on IgG PCs in peripheral lymphoid tissues. IgA PCs expressed antigen-presenting molecule MHC class II and co-stimulatory molecules CD80, CD86, and PD-L2. IgA PCs isolated from intestinal LP exhibited antigen presentation activity, and in the presence of TGF-β induced FoxP3(+) regulatory T cells, but not IFN-γ(+) Th1 cells, from naïve T cells. Thus, IgA PCs in the intestine may be involved in an immune regulatory role in the intestinal immune system.

to determine whether Treg defects resulted from either increased apoptosis or decreased maintenance of Foxp3 expression in Tregs.Methods/Results: WASP-deficient DCs led to decreased In Vitro generation of adaptive Tregs.More importantly, upon transfer of Foxp3cells into recipient mice, there was a marked reduction in the generation of Foxp3+ inducible Tregs in WASP/RAG DKO mice compared to RAG KO controls.The defects in WT Treg function in chimeric mice could not be explained by alterations in the maintenance or apoptosis of Foxp3+ donor cells in WASP/RAG DKO mice compared to RAG KO mice.Employing an In Vitro suppression assay, WT Tregs functioned normally when MLN DCs isolated from either WT or WASP-deficient mice were used as APCs.In contrast, there was aberrant Treg function when DCs were isolated from WASP/RAG DKO mice.Conclusions: WASP deficiency in innate immune cells leads to defective Treg generation and function In Vivo.Similarly, there was reduced generation of WT Tregs In Vitro in the presence of WASPdeficient DCs.However, Treg function was compromised only when DCs were harvested from WASP/RAG DKO mice but not WASP-deficient mice, suggesting an intrinsic aberrancy in WASP-deficient DCs unmasked in a lymphopenic setting.In summary, colitis in chimeric mice may result from DC-driven alterations in Treg number and function.

BACKGROUND & AIMS: Although T-helper (Th) 17 and Th1 cells are involved in pathogenesis of intestinal inflammation, their developmental pathways and sufficiency to promote disease are not known; nor are the roles of CD4(+)CD25(+) regulatory T (T(R)) cells in their development. METHODS: We performed adoptive transfer experiments to investigate the induction and suppression of colitis using naive CD4(+)CD45RB(high) T cells and or CD4(+)CD25(+) TR cells that were obtained from retinoid-related orphan receptor gamma t (ROR gamma t) gfp/(+) or Ly5.1/Ly5.2 congenic mice. RESULTS: We observed 3 types of colitogenic CD4(+) Th1 cells (interleukin [IL]-17A(-) interferon [IFN]-gamma(+)): ROR(gamma t-) classical Th1 cells that differentiated directly from naive T cells; ROR(gamma t+) Th1-like cells; and ROR(gamma t-) alternative Th1 cells that were terminally differentiated from ROR(gamma t+) cells via Th17 (IL-17A(+)IFN-gamma(-)), Th17 Th1 (IL-17A(+)IFN-gamma(+)), or Th1-like (IL-17A-IFN-gamma(+)) cells. In this pathway, CD4(+)CD25(+) TR cells suppress the development of not only classical Th1 cells, but also alternative Th1 cells at the transition of Th17 Th1 into alternative Th1 cells, resulting in accumulation of Th17 and Th17 Th1 cells in mice in which the development of colitis was suppressed. Furthermore, TR cells regulated the established balance of Th17 and Th1 cells under colitic conditions to yield a high ratio of Th17 and Th17 Th1 cells to Th1 cells in noncolitic conditions. CONCLUSIONS: Th17 and Th17 Th1 cells become colitogenic alternative Th1 cells via Th17, Th17 Th1, and Th1-like cells, independently of classical Th1 cells. TR cells suppress this pathway, resulting in accumulation of Th17 and Th17 Th1 cells.

Activation-induced cytidine deaminase (AID) is an essential factor for the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. CSR and SHM are initiated by AID-induced DNA breaks in the S and V regions, respectively. Because truncation or frame-shift mutations at the carboxyl (C)-terminus of AID abolishes CSR but not SHM, the C-terminal region of AID likely is required for the targeting of DNA breaks in the S region. To test this hypothesis, we determined the precise location and relative amounts of AID-induced DNA cleavage using an in situ DNA end-labeling method. We established CH12F3–2 cell transfectants expressing the estrogen receptor (ER) fused with wild-type (WT) AID or a deletion mutant lacking the C-terminal 16 aa, JP8Bdel. We found that AID-ER, but not JP8Bdel-ER, caused a CSR to IgA from the addition of 4-hydroxy tamoxifen. In contrast, both WT AID and JP8Bdel induced DNA breaks in both the V and S regions. In addition, JP8Bdel enhanced c-myc/IgH translocations. Our findings indicate that the C-terminal domain of AID is not required for S-region DNA breaks but is required for S-region recombination after DNA cleavage. Therefore, AID does not distinguish between the V and S regions for cleavage, but carries another function specific to CSR.

Activation-induced cytidine deaminase (AID) is the essential enzyme inducing the DNA cleavage required for both somatic hypermutation and class switch recombination (CSR) of the immunoglobulin gene. We originally proposed the RNA-editing model for the mechanism of DNA cleavage by AID. We obtained evidence that fulfils three requirements for CSR by this model, namely (i) AID shuttling between nucleus and cytoplasm, (ii) de novo protein synthesis for CSR, and (iii) AID–RNA complex formation. The alternative hypothesis, designated as the DNA-deamination model, assumes that the in vitro DNA deamination activity of AID is representative of its physiological function in vivo . Furthermore, the resulting dU was removed by uracil DNA glycosylase (UNG) to generate a basic site, followed by phosphodiester bond cleavage by AP endonuclease. We critically examined each of these provisional steps. We identified a cluster of mutants (H48A, L49A, R50A and N51A) that had particularly higher CSR activities than expected from their DNA deamination activities. The most striking was the N51A mutant that had no ability to deaminate DNA in vitro but retained approximately 50 per cent of the wild-type level of CSR activity. We also provide further evidence that UNG plays a non-canonical role in CSR, namely in the repair step of the DNA breaks. Taking these results together, we favour the RNA-editing model for the function of AID in CSR.