ZBP1 promotes hepatocyte pyroptosis in acute liver injury by regulating the PGAM5/ROS pathway

Introduction and Objectives: Acute liver injury (ALI) is characterized by massive hepatocyte death with high mortality and poor prognosis. Hepatocyte pyroptosis plays a key role in the physiopathological processes of ALI, which can damage mitochondria and release NLRP3 inflammasome particles, causing systemic inflammatory responses. Z-DNA Binding Protein 1 (ZBP1) is a sensor that induces cell death. Here, we investigated whether ZBP1 participates in hepatocyte pyroptosis and explored the possible pathogenesis of ALI. Materials and Methods: Hepatocyte pyrotosis was induced with lipopolysaccharide (LPS) and nigericin (Nig), and the expression of Zbp1 (ZBP1) was examined by western blot analysis and RT-qPCR. Further, we transfected AML-12 (LO2 and HepG2) cell lines with Zbp1 (ZBP1) siRNA. After ZBP1 was silenced, LDH release and flow cytometry were used to measure the cell death; Western blot analysis and RT-qPCR were used to detect the marker of NLRP3 inflammasome activation and pyroptosis. We also detected the expression of mitochondrial linear rupture marker phosphoglycerate mutase family member 5 (PGAM5) using western blot analysis and reactive oxygen species (ROS) using the DCFH-DA method. Results: The expression of ZBP1 was up-regulated in LPS/Nig-induced hepatocytes. Si-Zbp1 (Si-ZBP1) inhibited NLRP3 inflammasome activation and pyroptosis in LPS/Nig-induced hepatocytes. Moreover, ZBP1 silencing inhibited the expression of PGAM5 by reducing ROS production. Conclusions: ZBP1 promotes hepatocellular pyroptosis by modulating mitochondrial damage, which facilitates the extracellular release of ROS

The Novel Protein ADAMTS16 Promotes Gastric Carcinogenesis by Targeting IFI27 through the NF-κb Signaling Pathway

A disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16) has been reported to be involved in the pathogenesis of solid cancers. However, its role in gastric cancer (GC) is unclear. In this study, the role of ADAMTS16 in gastric cancer was investigated. The effects of ADAMTS16 on cell migration, invasion, and proliferation were investigated by functional experiments in vivo and in vitro. Downstream signal pathways of ADAMTS16 were confirmed by using bioinformatics analysis, co-immunoprecipitation, and immunofluorescence. Meanwhile, bioinformatics analysis, qRT-PCR, western blot, and dual-luciferase reporter gene analysis assays were used to identify ADAMTS16 targets. The expression of ADAMTS16 in GC was analyzed in public datasets. The expression of ADAMTS16 and its correlations with the clinical characteristics of GC were investigated by immunohistochemistry. Ectopic ADAMTS16 expression significantly promoted tumor cell migration, invasion, and growth. Bioinformatics analysis and western blot showed that ADAMTS16 upregulated the IFI27 protein through the NF-κb pathway, which was confirmed by immunofluorescence and western blot. Dual-luciferase reporter gene analysis identified a binding site between P65 and IFI27 that may be directly involved in the transcriptional regulation of IFI27. IFI27 knockdown reversed the promoting effect of ADAMTS16 on cell invasion, migration, and proliferation indicating that ADAMTS16 acts on GC cells by targeting the NF-κb/IFI27 axis. ADAMTS16 was associated with poor prognosis in clinical characteristics. ADAMTS16 promotes cell migration, invasion, and proliferation by targeting IFI27 through the NF-κB pathway and is a potential progressive and survival biomarker of GC

LAD1 promotes malignant progression by diminishing ubiquitin-dependent degradation of vimentin in gastric cancer

Abstract Background Ladinin-1 (LAD1), an anchoring filament protein, has been associated with several cancer types, including cancers of the colon, lungs, and breast. However, it is still unclear how and why LAD1 causes gastric cancer (GC). Methods Multiple in vitro and in vivo, functional gains and loss experiments were carried out in the current study to confirm the function of LAD1. Mass spectrometry was used to find the proteins that interact with LAD1. Immunoprecipitation analyses revealed the mechanism of LAD1 involved in promoting aggressiveness. Results The results revealed that the LAD1 was overexpressed in GC tissues, and participants with increased LAD1 expression exhibited poorer disease-free survival (DFS) and overall survival (OS). Functionally, LAD1 promotes cellular invasion, migration, proliferation, and chemoresistance in vivo and in vitro in the subcutaneous patient-and cell-derived xenograft (PDX and CDX) tumor models. Mechanistically, LAD1 competitively bound to Vimentin, preventing it from interacting with the E3 ubiquitin ligase macrophage erythroblast attacher (MAEA), which led to a reduction in K48-linked ubiquitination of Vimentin and an increase in Vimentin protein levels in GC cells. Conclusions In conclusion, the current investigation indicated that LAD1 has been predicted as a possible prognostic biomarker and therapeutic target for GC due to its ability to suppress Vimentin–MAEA interaction

Intercellular adhesion molecule 2 as a novel prospective tumor suppressor induced by ERG promotes ubiquitination-mediated radixin degradation to inhibit gastric cancer tumorigenicity and metastasis

Abstract Background Gastric cancer (GC) is a fatal cancer with unclear pathogenesis. In this study, we explored the function and potential mechanisms of intercellular adhesion molecule 2 (ICAM2) in the development and advancement of GC. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to quantify ICAM2 expression in harvested GC tissues and cultured cell lines. Immunohistochemical analyses were conducted on a GC tissue microarray to quantify ICAM2 expression and explore its implication on the prognosis of GC patients. In vitro experiments were carried out to reveal the biological functions of ICAM2 in GC cell lines. Further, in vivo experiments were conducted using xenograft models to assess the impact of ICAM2 on GC development and metastasis. Western blot, immunofluorescence, immunoprecipitation, luciferase assay, chromatin immunoprecipitation, and ubiquitination analysis were employed to investigate the underlying mechanisms. Results ICAM2 expression was downregulated in GC, positively correlating with advanced T stage, distant metastasis, advanced clinical stage, vessel invasion, and shorter patient survival time. ICAM2 overexpression suppressed the proliferation, migration, invasion, metastasis of GC cells as well as their ability to form tumors, whereas ICAM2 knockdown yielded opposite results. Erythroblast transformation-specific-related gene (ERG) as a transcription factor promoted the transcription of ICAM2 by binding to the crucial response element localized within its promoter region. Further analysis revealed that ICAM2 reduced radixin (RDX) protein stability and expression. In these cells, ICAM2 bound to the RDX protein to promote the ubiquitination and degradation of RDX via NEDD4 Like E3 Ubiquitin Protein Ligase (NEDD4L), and this post-translational modification resulted in the inhibition of GC. Conclusions In summary, this study demonstrates that ICAM2, which is induced by ERG, suppresses GC progression by enhancing the ubiquitination and degradation of RDX in a NEDD4L-dependent manner. Therefore, these results suggest that ICAM2 is a potential prognostic marker and a therapeutic target for GC

Induction of filopodia formation by α-Actinin-2 via RelA with a feedforward activation loop promoting overt bone marrow metastasis of gastric cancer

Abstract Background Bone marrow metastasis (BMM) is underestimated in gastric cancer (GC). GC with BMM frequently complicate critical hematological abnormalities like diffused intravascular coagulation and microangiopathic hemolytic anemia, which constitute a highly aggressive GC (HAGC) subtype. HAGC present a very poor prognosis with peculiar clinical and pathological features when compared with not otherwise specified advanced GC (NAGC). But the molecular mechanisms underlying BMM from GC remain rudimentary. Methods The transcriptomic difference between HAGC and NAGC were analyzed. Genes that were specifically upregulated in HAGC were identified, and their effect on cell migration and invasion was studied. The function of ACTN2 gene were confirmed by GC cell lines, bone-metastatic animal model and patients’ tissues. Furthermore, the molecular mechanism of ACTN2 derived-BMM was explored by multiple immunofluorescence staining, western blot, chromatin immunoprecipitation, and luciferase reporter assays. Results We elucidated the key mechanisms of BMM depending on the transcriptomic difference between HAGC and NAGC. Five genes specifically upregulated in HAGC were assessed their effect on cell migration and invasion. The ACTN2 gene encoding protein α-Actinin-2 was detected enhanced the metastatic capability and induced BMM of GC cells in mouse models. Mechanically, α-Actinin-2 was involved in filopodia formation where it promoted the Actin filament cross-linking by replacing α-Actinin-1 to form α-Actinin-2:α-Actinin-4 complexes in GC cells. Moreover, NF-κB subunit RelA and α-Actinin-2 formed heterotrimers in the nuclei of GC cells. As a direct target of RelA:α-Actinin-2 heterotrimers, the ACTN2 gene was a positive auto-regulatory loop for α-Actinin-2 expression. Conclusions We demonstrated a link between filopodia, BMM and ACTN2 activation, where a feedforward activation loop between ACTN2 and RelA is established via actin in response to distant metastasis. Given the novel filopodia formation function and the new mechanism of BMM in GC, we propose ACTN2 as a druggable molecular vulnerability that may provide potential therapeutic benefit against BMM of GC

Additional file 2 of Intercellular adhesion molecule 2 as a novel prospective tumor suppressor induced by ERG promotes ubiquitination-mediated radixin degradation to inhibit gastric cancer tumorigenicity and metastasis

Additional file 2. The predicted E3 ubiquitin ligases of RDX. (a) UbiBrowser was used to predict the E3 ubiquitin ligases of the RDX. (b) RT-PCR was performed to verify the knockdown efficiency of the main E3 ubiquitin ligases in GC cells

Additional file 1 of Intercellular adhesion molecule 2 as a novel prospective tumor suppressor induced by ERG promotes ubiquitination-mediated radixin degradation to inhibit gastric cancer tumorigenicity and metastasis

Additional file 1. ICAM2 plays tumour‐suppressive roles in GC cell. (a) The efficiency ICAM2 overexpression or knockdown were confirmed by RT-PCR. (b) Flow cytometry analysis of the effect of ICAM2 overexpression or knockdown on the cell cycle progression of GC cells. (c) Flow cytometry results showing the effect of ICAM2 overexpression or knockdown on the apoptosis of GC cells. (d) Representative image of the wound healing assays

Additional file 3 of Intercellular adhesion molecule 2 as a novel prospective tumor suppressor induced by ERG promotes ubiquitination-mediated radixin degradation to inhibit gastric cancer tumorigenicity and metastasis

Additional file 3. Restoration of RDX reverses the antitumor effect of ICAM2 overexpression in GC. (a) The efficiency of RDX overexpression was confirmed by RT-PCR. (b) Representative image of the wound healing assays. (c) The apoptosis of ICAM2-overexpressing cells treated with RDX was confirmed by flow cytometry. (d) Transfection efficiency after overexpression of RDX in GC cells was determined by western blot