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.
Subject(s)
Adenocarcinoma of Lung , Caspases, Initiator , Interferon-gamma , Lung Neoplasms , Pyroptosis , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/pathology , Adenocarcinoma of Lung/metabolism , Animals , Interferon-gamma/metabolism , Interferon-gamma/pharmacology , Interferon-gamma/genetics , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Mice , Caspases, Initiator/metabolism , Caspases, Initiator/genetics , Cell Line, Tumor , Neoplasm Metastasis , Gene Expression Regulation, NeoplasticABSTRACT
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.
Subject(s)
Actin Depolymerizing Factors , Actins , Actins/metabolism , Actin Depolymerizing Factors/metabolism , Cell Movement , Mitosis , Epithelial Cells/metabolism , Lim Kinases/genetics , PhosphorylationABSTRACT
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.
Subject(s)
Angiopoietin-Like Protein 8 , Carcinoma, Renal Cell , Kidney Neoplasms , Peptide Hormones , Humans , Angiopoietin-Like Protein 8/genetics , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Cell Differentiation , Inflammation , Kidney Neoplasms/genetics , Peptide Hormones/metabolism , Tumor MicroenvironmentABSTRACT
Wnt/ß-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 ß-catenin/CBP but not for ß-catenin/p300. By taking advantage of the action of ICG-001, we sought to investigate regulatory mechanisms underlying ß-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 ß-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 ß-catenin coactivator usage in PANC-1 cells. These results would further our understanding of the canonical Wnt/ß-catenin signaling divergence.
ABSTRACT
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 α5ß1, α6, and ß4, but not those of αvß3, α3, α4, and ß1, 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.
Subject(s)
Angiopoietin-like Proteins/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Peritoneal Neoplasms/metabolism , Peritoneal Neoplasms/secondary , Angiopoietin-Like Protein 2 , Animals , Anoikis/physiology , Cell Line, Tumor , Cell Proliferation/physiology , Female , Heterografts , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Signal TransductionABSTRACT
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.
Subject(s)
B7-H1 Antigen/immunology , Carcinoma, Non-Small-Cell Lung/immunology , Lung Neoplasms/immunology , Programmed Cell Death 1 Receptor/immunology , Antibodies/immunology , B7-H1 Antigen/blood , B7-H1 Antigen/metabolism , Blotting, Western , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Glycosylation , Humans , Lung Neoplasms/blood , Lung Neoplasms/metabolism , Programmed Cell Death 1 Receptor/blood , Programmed Cell Death 1 Receptor/metabolism , Protein Binding , Protein Transport , SolubilityABSTRACT
Lung cancer cells, particularly those of non-small-cell lung cancer, are known to express Nectin-4. We previously generated a recombinant measles virus that uses Nectin-4 as its receptor but cannot bind its original principal receptor, signaling lymphocyte activation molecule (SLAM). This virus (rMV-SLAMblind) infects and kills breast cancer cells in vitro and in a subcutaneous xenograft model. However, it has yet to be determined whether rMV-SLAMblind is effective against other cancer types and in other tumor models that more closely represent disease. In this study, we analyzed the anti-tumor activity of this virus towards lung cancer cells using a modified variant that encodes green fluorescent protein (rMV-EGFP-SLAMblind). We found that rMV-EGFP-SLAMblind efficiently infected nine, human, lung cancer cell lines, and its infection resulted in reduced cell viability of six cell lines. Administration of the virus into subcutaneous tumors of xenotransplanted mice suppressed tumor growth. In addition, rMV-EGFP-SLAMblind could target scattered tumor masses grown in the lungs of xenotransplanted mice. These results suggest that rMV-SLAMblind is oncolytic for lung cancer and that it represents a promising tool for the treatment of this disease.
Subject(s)
Carcinoma, Non-Small-Cell Lung/therapy , Lung Neoplasms/therapy , Measles virus/physiology , Oncolytic Virotherapy/methods , Oncolytic Viruses/physiology , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/virology , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Female , Flow Cytometry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Lung Neoplasms/genetics , Lung Neoplasms/virology , Measles virus/genetics , Measles virus/metabolism , Mice, SCID , Microscopy, Fluorescence , Oncolytic Viruses/genetics , Oncolytic Viruses/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Signaling Lymphocytic Activation Molecule Family Member 1 , Xenograft Model Antitumor AssaysABSTRACT
The production of IgE, a main player in allergic disorders such as asthma and atopic dermatitis, is strictly regulated and the serum concentrations of IgE are normally kept at a much lower level than other isotypes. We found that mice deficient for the p85alpha regulatory subunit of class IA phosphoinositide 3-kinase (PI3K) produced increasing amounts of serum IgE. Purified p85alpha-/- B cells produced more IgE than wild-type B cells in vitro in response to anti-CD40 mAb and IL-4. PI3K inhibitors wortmannin and IC87114 enhanced IgE production by wild-type B cells stimulated with anti-CD40 mAb and IL-4. Under the same condition, antigen receptor cross-linking induced the expression of inhibitor of differentiation-2 and suppressed the expression of activation-induced cytidine deaminase and class switch recombination (CSR) in a PI3K-dependent manner. IgE production was also suppressed in a concentrated cell culture condition, which was completely reversed by PI3K inhibition. The selective suppression of IgE production by PI3K was also observed at a protein level after CSR. Our results indicate that PI3K negatively regulates IgE production at both CSR and protein levels.
Subject(s)
Immunoglobulin E/biosynthesis , Phosphatidylinositol 3-Kinases/immunology , Animals , Antibodies/immunology , Antigen-Antibody Reactions , B-Lymphocytes/immunology , Cells, Cultured , Cytidine Deaminase/antagonists & inhibitors , Cytidine Deaminase/genetics , Cytidine Deaminase/immunology , Down-Regulation , Gene Expression Profiling , Immunoglobulin Class Switching/drug effects , Immunoglobulin Class Switching/genetics , Immunoglobulin Class Switching/immunology , Immunoglobulin E/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphatidylinositol 3-Kinases/deficiency , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Ig class switch recombination (CSR) is triggered by the engagement of CD40 on B cells by CD40 ligand on T cells. In addition, recent studies have shown that dendritic cells (DCs) are able to directly control the CSR of B cells through B lymphocyte stimulator protein [or B cell activation factor belonging to the tumor necrosis factor family] and a proliferation-inducing ligand. We examined in this study the regulatory role of DCs in CSR and demonstrate that DCs selectively suppress IgE production from B cells stimulated by CD40 and IL-4 through two different mechanisms: by direct cell-cell interaction or by soluble factors including transforming growth factor-beta and IFN-gamma. In addition, distinct DCs utilize different mechanisms: immature bone marrow-derived dendritic cells (BMDCs) and primary lung DCs strongly inhibit IgE CSR. On the other hand, LPS-induced mature BMDCs lose the ability to inhibit IgE CSR but still suppress IgE production by decreasing IgE protein expression. These results indicate novel regulatory functions of DCs on IgE production.