ADAR1 masks the cancer immunotherapeutic promise of ZBP1-driven necroptosis.

Only a small proportion of patients with cancer show lasting responses to immune checkpoint blockade (ICB)-based monotherapies. The RNA-editing enzyme ADAR1 is an emerging determinant of resistance to ICB therapy and prevents ICB responsiveness by repressing immunogenic double-stranded RNAs (dsRNAs), such as those arising from the dysregulated expression of endogenous retroviral elements (EREs)1-4. These dsRNAs trigger an interferon-dependent antitumour response by activating A-form dsRNA (A-RNA)-sensing proteins such as MDA-5 and PKR5. Here we show that ADAR1 also prevents the accrual of endogenous Z-form dsRNA elements (Z-RNAs), which were enriched in the 3' untranslated regions of interferon-stimulated mRNAs. Depletion or mutation of ADAR1 resulted in Z-RNA accumulation and activation of the Z-RNA sensor ZBP1, which culminated in RIPK3-mediated necroptosis. As no clinically viable ADAR1 inhibitors currently exist, we searched for a compound that can override the requirement for ADAR1 inhibition and directly activate ZBP1. We identified a small molecule, the curaxin CBL0137, which potently activates ZBP1 by triggering Z-DNA formation in cells. CBL0137 induced ZBP1-dependent necroptosis in cancer-associated fibroblasts and reversed ICB unresponsiveness in mouse models of melanoma. Collectively, these results demonstrate that ADAR1 represses endogenous Z-RNAs and identifies ZBP1-mediated necroptosis as a new determinant of tumour immunogenicity masked by ADAR1. Therapeutic activation of ZBP1-induced necroptosis provides a readily translatable avenue for rekindling the immune responsiveness of ICB-resistant human cancers.

Effects of smokeless tobacco on cell viability, reactive oxygen species, apoptosis, and inflammatory cytokines in human umbilical vein endothelial cells.

Smokeless tobacco products provide an alternative to cigarettes; however, smokeless tobacco is carcinogenic and harmful to human health. This study evaluated the toxicological effects of snus extracts and cigarette smoke total particulate matter (TPM) on human umbilical vein endothelial cells (HUVECs). Treated cells were examined for cell viability, reactive oxygen species (ROS), apoptosis, and inflammatory cytokines. Moreover, we explored the mechanism of programmed cell death induced by snus. The results showed that snus extracts significantly inhibited cell viability in a dose-dependent manner. ROS was significantly increased in treatment groups, and anti-oxidant treatment could not prevent snus extract-induced cell death. Snus extracts induced apoptosis, DNA damage, activation and cleavage of caspase-3 and caspase-8, pathway-related gene change, and interleukin (IL)-6 and IL-8 release in HUVECs. Snus extracts exposure may induce cytotoxicity, ROS generation, inflammatory cytokines release, and apoptosis or DNA damage through intrinsic and extrinsic pathways in HUVECs.

Analysis of methyl DNA adducts and metabolites in BEAS-2B cells induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is classified as a Group 1 human carcinogen. It is metabolically activated by P450 enzymes to intermediate methylate and pyridyloxobutylate DNA, resulting in the formation of DNA adduct that is critical for the carcinogenicity of NNK. To directly and objectively examine the DNA adduct formation profiles without the complexity of factors in vivo, in the present study, five kinds of methyl DNA adducts were first identified in the incubation model of NNK established with human lung epithelial cells (BEAS-2B). The level of methyl DNA adducts and metabolites of NNK were quantitatively analyzed, respectively. With the increase of exposure time and dose, the level of methyl DNA adducts and metabolites increased. Furthermore, with the changes of the activity of P450 enzymes, which is the main enzyme regulating the α-hydroxylation of NNK, we found the levels of both methyl adducts and metabolites formed via α-hydroxylation in experimental groups showed the same trend compared with those in control group, while the metabolites formed via other pathways changed in the opposite trend. The result proves that the methyl adducts induced by NNK generate via α-hydroxylation pathway in BEAS-2B cells.

Stat3-Efemp2a modulates the fibrillar matrix for cohesive movement of prechordal plate progenitors.

Recently, emerging evidence has shown that Stat3 controls tumor cell migration and invasion. However, the molecular mechanisms by which Stat3 controls the cell movement remain largely unknown. Embryonic gastrula progenitors display coordinated and orientated migration, called collective cell migration. Collective cell migration is the simultaneous movement of multiple cells and is universally involved in physiological and pathological programs. Stat3 activity is required for the migration of gastrula progenitors, but it does not affect cell specification, thus suggesting that gastrula movements are an excellent model to provide insight into Stat3 control of cell migration in vivo. In this study, we reveal a novel mechanism by which Stat3 modulates extracellular matrix (ECM) assembly to control the coherence of collective migration of prechordal plate progenitors during zebrafish embryonic gastrulation. We show that Stat3 regulates the expression of Efemp2a in the prechordal plate progenitors that migrate anteriorly during gastrulation. Alteration of Stat3-Efemp2a signaling activity disrupted the configuration of fibronectin (FN) and laminin (LM) matrices, resulting in defective coherence of prechordal plate progenitor movements in zebrafish embryos. We demonstrate that Efemp2a acts as a downstream effector of Stat3 to promote ECM configuration for coherent collective cell migrations in vivo.

Zα domain proteins mediate the immune response.

The Zα domain has a compact α/ß architecture containing a three-helix bundle flanked on one side by a twisted antiparallel ß sheet. This domain displays a specific affinity for double-stranded nucleic acids that adopt a left-handed helical conformation. Currently, only three Zα-domain proteins have been identified in eukaryotes, specifically ADAR1, ZBP1, and PKZ. ADAR1 is a double-stranded RNA (dsRNA) binding protein that catalyzes the conversion of adenosine residues to inosine, resulting in changes in RNA structure, function, and expression. In addition to its editing function, ADAR1 has been shown to play a role in antiviral defense, gene regulation, and cellular differentiation. Dysregulation of ADAR1 expression and activity has been associated with various disease states, including cancer, autoimmune disorders, and neurological disorders. As a sensing molecule, ZBP1 exhibits the ability to recognize nucleic acids with a left-handed conformation. ZBP1 harbors a RIP homotypic interaction motif (RHIM), composed of a highly charged surface region and a leucine-rich hydrophobic core, enabling the formation of homotypic interactions between proteins with similar structure. Upon activation, ZBP1 initiates a downstream signaling cascade leading to programmed cell death, a process mediated by RIPK3 via the RHIM motif. PKZ was identified in fish, and contains two Zα domains at the N-terminus. PKZ is essential for normal growth and development and may contribute to the regulation of immune system function in fish. Interestingly, some pathogenic microorganisms also encode Zα domain proteins, such as, Vaccinia virus and Cyprinid Herpesvirus. Zα domain proteins derived from pathogenic microorganisms have been demonstrated to be pivotal contributors in impeding the host immune response and promoting virus replication and spread. This review focuses on the mammalian Zα domain proteins: ADAR1 and ZBP1, and thoroughly elucidates their functions in the immune response.

PLEKHG5 is stabilized by HDAC2-related deacetylation and confers sorafenib resistance in hepatocellular carcinoma.

Sorafenib is the first FDA-approved first-line targeted drug for advanced HCC. However, resistance to sorafenib is frequently observed in clinical practice, and the molecular mechanism remains largely unknown. Here, we found that PLEKHG5 (pleckstrin homology and RhoGEF domain containing G5), a RhoGEF, was highly upregulated in sorafenib-resistant cells. PLEKHG5 overexpression activated Rac1/AKT/NF-κB signaling and reduced sensitivity to sorafenib in HCC cells, while knockdown of PLEKHG5 increased sorafenib sensitivity. The increased PLEKHG5 was related to its acetylation level and protein stability. Histone deacetylase 2 (HDAC2) was found to directly interact with PLEKHG5 to deacetylate its lysine sites within the PH domain and consequently maintain its stability. Moreover, knockout of HDAC2 (HDAC2 KO) or selective HDAC2 inhibition reduced PLEKHG5 protein levels and thereby enhanced the sensitivity of HCC to sorafenib in vitro and in vivo, while overexpression of PLEKHG5 in HDAC2 KO cells reduced the sensitivity to sorafenib. Our work showed a novel mechanism: HDAC2-mediated PLEKHG5 posttranslational modification maintains sorafenib resistance. This is a proof-of-concept study on targeting HDAC2 and PLEKHG5 in sorafenib-treated HCC patients as a new pharmaceutical intervention for advanced HCC.

Cigarette smoke-induced oxidative stress activates NRF2 to mediate fibronectin disorganization in vascular formation.

Cigarette smoke significantly induces oxidative stress, resulting in cardiovascular disease. NRF2, a well-known antioxidative stress response factor, is generally considered to play protective roles in cardiovascular dysfunction triggered by oxidative stress. Interestingly, recent studies reported adverse effects of NRF2 on the cardiovascular system. These unfavourable pathogenic effects of NRF2 need to be further investigated. Our work shows that cigarette smoke extract (CSE)-induced oxidative stress disturbs fibronectin (FN) assembly during angiogenesis. Furthermore, this effect largely depends on hyperactive NRF2-STAT3 signalling, which consequently promotes abnormal FN deposition. Consistently, disruption of this pathway by inhibiting NRF2 or STAT3 prevents CSE-induced FN disorganization and vasculature disruption in human umbilical vein endothelial cells or zebrafish. Taken together, these findings demonstrate the cardiovascular dysfunction caused by CSE from a novel perspective that NRF2-dependent signalling engages in FN disorganization.

Establishment of rapid risk assessment model for cigarette smoke extract exposure in chronic obstructive pulmonary disease.

Rapid risk assessment models for different types of cigarette smoke extract (CSE) exposure are critical to understanding the etiology of chronic obstructive pulmonary disease. The present study investigated inflammation of cultured tracheal tissues with CSE exposure. Rat trachea rings were isolated, cultured, then exposed to various concentrations of CSE from 3R4 F reference cigarettes for 4 h. Tissue/cellular morphology, ultrastructure, viability and damage, inflammatory cell infiltration, and inflammatory protein levels were measured and compared to untreated controls. Human bronchial epithelial cells (BEAS-2B) exposed to 0 or 300 µg/mL CSE were cocultured with macrophages to assess extent of mobilization and phagocytosis. Endotracheal epithelium cilia densities were significantly reduced with increasing CSE concentrations, while mucous membranes became increasingly disordered; both eventually disappeared. Macrophages became larger as the CSE concentration increased, with microvilli and extended pseudopodium covering their surface, and many primary and secondary lysosomes present in the cytoplasm. Inflammatory cell infiltration also increased with increasing CSE dose, as did intracellular adhesion molecule-1(ICAM-1), interleukin-6(IL-6). The method described here may be useful to qualitatively characterized the effects of the compound under study. Then, we use BEAS-2B cell line system to strength the observation made in the cultured tissues. Probably, an approach to integrate results from both experiments will facilitate its application. These results demonstrate that cultured rat tracheal rings have a whole-tissue structure that undergoes inflammatory processes similar to in vivo tissues upon CSE exposure.

Syndecan-4 modulates the proliferation of neural cells and the formation of CaP axons during zebrafish embryonic neurogenesis.

Syndecan-4 (Syn4), a single-pass transmembrane heparin sulphate proteoglycan (HSPG), plays significant role in the formation of focal adhesions and interacts with many growth factors to regulate cell migration and neural induction. Here, we show the new roles of syndecan-4(syn4) in zebrafish embryonic neurogenesis. Syn4 is broadly and dynamically expressed throughout the early stages of embryonic development. Knockdown of syn4 increases the expression of the marker genes of multiple types of neural cells. The increased expression of the marker genes is resulted from excessive proliferation of the neural cells. In addition, disrupting syn4 expression results in truncated and multiple aberrant branching of caudal primary (CaP) axons. Collectively, these data indicate that Syn4 suppresses the cellular proliferation during neurogenesis and is crucial for the formation of CaP axons during zebrafish embryogenesis.

Snail modulates the assembly of fibronectin via α5 integrin for myocardial migration in zebrafish embryos.

The Snail family member snail encodes a zinc finger-containing transcriptional factor that is involved in heart formation. Yet, little is known about how Snail regulates heart development. Here, we identified that one of the duplicated snail genes, snai1b, was expressed in the heart region of zebrafish embryos. Depletion of Snai1b function dramatically reduced expression of α5 integrin, disrupted Fibronectin layer in the heart region, especially at the midline, and prevented migration of cardiac precursors, resulting in defects in cardiac morphology and function in zebrafish embryos. Injection of α5ß1 protein rescued the Fibronectin layer and then the myocardial precursor migration in snai1b knockdown embryos. The results provide the molecular mechanism how Snail controls the morphogenesis of heart during embryonic development.

TLR7-expressing cells comprise an interfollicular epidermal stem cell population in murine epidermis.

Normal interfollicular epidermis (IFE) homeostasis is maintained throughout the entire life by its own stem cells that self-renew and generate progeny that undergo terminal differentiation. However, the fine markers of the stem cells in interfollicular epidermis are not well defined yet. Here we found that TLR7 identified the existence of progenitors and interfollicular epidermal stem cells in murine skin. In vitro, TLR7-expressing cells comprised of two subpopulations that were competent to proliferate and exhibited distinct differentiation potentials. Three-dimensional (3D) organotypic culture and skin reconstitution assays showed that TLR7-expressing cells were able to reconstruct the interfollicular epidermis. Finally, TLR7-expressing cells maintained the intact interfollicular epidermal structures revealed in serial transplantation assays in vivo in mice. Taken together, our results suggest that TLR7-expressing cells comprise an interfollicular epidermal stem cell population.