Arsenic and the compounds thereof can be carcinogens or therapeutic agents for different cancer types. However, for breast cancer (BC), studies have yielded conflicted results on the role of arsenic. A previous study by the present authors indicated a potential relationship between circDHX34 and sodium arsenite-treated BC cells. As such, the expression, function, and potential mechanism of circDHX34 in sodium arsenite-treated MDA-MB-231 cells were further detected. In the present study, findings were made that sodium arsenite upregulated circDHX34 expression in MDA-MB-231 cells in a dose-dependent manner, and knockdown of circDHX34 could promote cell proliferation and inhibit apoptosis. Further investigations revealed that knockdown of circDHX34 upregulated the expression levels of antiapoptotic genes BCL2 and BCL2L1 and downregulated the expression levels of proapoptotic genes CASP8 and CASP9. To conclude, by regulating apoptotic genes, sodium arsenite-mediated upregulation of circDHX34 promotes apoptosis in hormone-independent breast cancer cells.

Keywords: Apoptosis; Breast cancer; Circular RNA; Sodium arsenite.

Background: Idiopathic pulmonary fibrosis (IPF) is disease with high mortality, and its effective treatment is limited. Shenfu injection is a traditional Chinese medicine which can improve circulation and protect cells. In this study, we aimed to investigate the feasibility and mechanism of Shenfu injection in the treatment of IPF. Methods: The components and targets of Shenfu injection were mainly retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database. The targets of Shenfu injection were standardized by UniProt database. The Genecards and OMIM databases was used to search for IPF-related genes. The Venn diagram of gene intersection was drawn using the OmicStudio tools, and the protein-protein interaction network was visualized using the Cytoscape 3.7.2 software. Moreover, the metascape online software was applied to explore the enriched Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and the Cytoscape 3.7.2 software was used to construct the target-pathway network. Molecular docking was used to visualize the interactions between the main active compounds and targeted proteins. Animal experiments were performed to validate the effects and mechanisms of Shenfu injection. Results: We obtained 46 co-targets of Shenfu injection and IPF. Among the hub target genes, several genes with important functions were enriched, including TNF, IL-6, IL-1B, TP53, JUN, CASP3 and CASP8. The pathway enrichment analysis for the hub target genes identified pathways in infection/inflammation, apoptosis and cancer. Molecular docking results showed that the main active compound Ginsenoside Re had high affinity to the core target proteins. These results suggested that Shenfu injection may have a positive effect in the treatment of IPF. Consistent with this finding, animal experiments showed that Shenfu injection significantly reduced pulmonary fibrosis in a mouse model with inhibition of apoptosis and inflammation by downregulating IL-1β, caspase-3 and phosphorylated NF-κB. Conclusion: Our results demonstrated that Shenfu injection efficiently alleviate bleomycin-induced pulmonary fibrosis through multi-targets in inflammation-, apoptosis- and cancer-related pathways, which provided first evidence and reference to the feasibility of Shenfu injection in the treatment of IPF.

Keywords: Shenfu injection; apoptosis; idiopathic pulmonary fibrosis; inflammation; network pharmacology.

Background: Recently, mesenchymal stem cells therapy has been performed in dogs, although the outcome is not always favorable.

Objectives: To investigate the therapeutic efficacy of mesenchymal stem cells (MSCs) using dog leukocyte antigen (DLA) matching between the donor and recipient in vitro.

Methods: Canine adipose-derived MSCs (cA-MSCs) isolated from the subcutaneous tissue of Dog 1 underwent characterization. For major DLA genotyping (DQA1, DQB1, and DRB1), peripheral blood mononuclear cells (PBMCs) from two dogs (Dogs 1 and 2) were analyzed by direct sequencing of polymerase chain reaction (PCR) products. The cA-MSCs were co-cultured at a 1:10 ratio with activated PBMCs (DLA matching or mismatching) for 3 days and analyzed for immunosuppressive (IDO, PTGS2, and PTGES), inflammatory (IL6 and IL10), and apoptotic genes (CASP8, BAX, TP53, and BCL2) by quantitative real-time reverse transcriptase-PCR.

Results: cA-MSCs were expressed cell surface markers such as CD90⁺/44⁺/29⁺/45⁻ and differentiated into osteocytes, chondrocytes, and adipocytes in vitro. According to the Immuno Polymorphism Database, DLA genotyping comparisons of Dogs 1 and 2 revealed complete differences in genes DQA1, DQB1, and DRB1. In the co-culturing of cA-MSCs and PBMCs, DLA mismatch between the two cell types induced a significant increase in the expression of immunosuppressive (IDO/PTGS2) and apoptotic (CASP8/BAX) genes.

Conclusions: The administration of cA-MSCs matching the recipient DLA type can alleviate the need to regulate excessive immunosuppressive responses associated with genes, such as IDO and PTGES. Furthermore, easy and reliable DLA genotyping technology is required because of the high degree of genetic polymorphisms of DQA1, DQB1, and DRB1 and the low readability of DLA 88.

Keywords: Dog; adipose-derived mesenchymal stem cells; allogeneic transplantation; immunosuppressive; leukocyte antigen.

The methylation of RNA at the N6 position of adenosine (m⁶A) orchestrates multiple biological processes to control development, differentiation, and cell cycle, as well as various aspects of the virus life cycle. How the m⁶A RNA modification pathway is regulated to finely tune these processes remains poorly understood. Here, we discovered the m⁶A reader YTHDF2 as a caspase substrate via proteome-wide prediction, followed by in vitro and in vivo validations. We further demonstrated that cleavage-resistant YTHDF2 blocks, while cleavage-mimicking YTHDF2 fragments promote, the replication of a common human oncogenic virus, Epstein-Barr virus (EBV). Intriguingly, our study revealed a feedback regulation between YTHDF2 and caspase-8 via m⁶A modification of CASP8 mRNA and YTHDF2 cleavage during EBV replication. Further, we discovered that caspases cleave multiple components within the m⁶A RNA modification pathway to benefit EBV replication. Our study establishes that caspase disarming of the m⁶A RNA modification machinery fosters EBV replication. IMPORTANCE The discovery of an N⁶-methyladenosine (m⁶A) RNA modification pathway has fundamentally altered our understanding of the central dogma of molecular biology. This pathway is controlled by methyltransferases (writers), demethylases (erasers), and specific m⁶A binding proteins (readers). Emerging studies have linked the m⁶A RNA modification pathway to the life cycle of various viruses. However, very little is known regarding how this pathway is subverted to benefit viral replication. In this study, we established an unexpected linkage between cellular caspases and the m⁶A modification pathway, which is critical to drive the reactivation of a common tumor virus, Epstein-Barr virus (EBV).

Keywords: Epstein-Barr virus; METTL14; METTL3; WTAP; YTHDF2; caspase cleavage; lytic replication; m6A RNA modification; reactivation; restriction factor.

Oral squamous cell carcinoma (OSCC) is the most common type of oral malignancy. Our study uses multipoint materials to explore the heterogeneity and metastasis mechanism of OSCC to find more accurate molecular markers and new therapeutic targets. By using whole-exome capture and sequencing and tumor evolution analysis, we found that most clone-driven mutations were located in the branches of tumor phylogenetic tree, such as COTL1, CASP8, and PROCR. Most clone-driven OSCC mutations occur mainly in tumor suppressor genes, including TP53, SFRP4, and NOTCH1. Our study on intratumor heterogeneity (ITH) and clonal evolution provides an important molecular basis for further understanding of OSCC occurrence and development and metastasis and provides potential targets for the treatment of this disease.

Keywords: cancer biology; cell signalling; clone evolution biomarkers; heterogeneity; molecular genetics; oral carcinogenesis; oral squamous cell carcinoma (OSCC).

Glioma is the most common malignant primary brain tumour. It is of great significance for the prognosis and personalized treatment of glioma patients to accurate identification of glioma based on biomarkers. Pyroptosis, a kind of programmed cell death, is closely related to tumour progression and tumour immune microenvironment. However, the role of pyroptosis in glioma remained unclear. Herein, we used glioma clinical and expression data from TCGA and CGGA to explore the relationship between pyroptosis and glioma. We first summarized the incidence of copy number variations and somatic mutations of 33 pyroptosis-related genes and explored prognostic correlation of these genes. Based on pyroptosis-related genes, three molecular subgroups of glioma related to prognosis were identified. We also found that each subgroup has unique immune and biological behaviours characteristics. Finally, based on 7 pyroptosis-related genes (CASP3, CASP4, CASP6, CASP8, CASP9, PRKACA and ELANE), we constructed a prognosis model by Lasso and Cox regression, which had a strong predictive power for the overall survival in CGGA test cohort (p < 0.05). In summary, we explored the role of pyroptosis-related genes in gliomas and the association of these genes with tumour immunity. We found the biomarkers valuable to diagnosis and prognosis, hence, provide reference to the development and treatment of tumorigenesis in glioma.

Keywords: glioma; immunity; prognosis; pyroptosis.

To enable long-term survival, mammalian adult neurons exhibit unique apoptosis competence. Questions remain as to whether and how neurons globally reprogram the expression of apoptotic genes during development. We systematically examined the in vivo expression of 1923 apoptosis-related genes and associated histone modifications at eight developmental ages of mouse brains. Most apoptotic genes displayed consistent temporal patterns across the forebrain, midbrain, and hindbrain, suggesting ubiquitous robust developmental reprogramming. Although both anti- and pro-apoptotic genes can be up- or downregulated, half the regulatory events in the classical apoptosis pathway are downregulation of pro-apoptotic genes. Reduced expression in initiator caspases, apoptosome, and pro-apoptotic Bcl-2 family members restrains effector caspase activation and attenuates neuronal apoptosis. The developmental downregulation of apoptotic genes is attributed to decreasing histone-3-lysine-4-trimethylation (H3K4me3) signals at promoters, where histone-3-lysine-27-trimethylation (H3K27me3) rarely changes. By contrast, repressive H3K27me3 marks are lost in the upregulated gene groups, for which developmental H3K4me3 changes are not predictive. Hence, developing brains remove epigenetic H3K4me3 and H3K27me3 marks on different apoptotic gene groups, contributing to their downregulation and upregulation, respectively. As such, neurons drastically alter global apoptotic gene expression during development to transform apoptosis controls. Research into neuronal cell death should consider maturation stages as a biological variable.

Keywords: Apaf-1; Bak1; Bax; Casp3; Casp6; Casp7; Casp8; Casp9; cytochrome c; epigenome.

Background: Prenatal exposure to essential and non-essential metals impacts birth and child health, including fetal growth and neurodevelopment. DNA methylation (DNAm) may be involved in pathways linking prenatal metal exposure and health. In the Project Viva cohort, we analyzed the extent to which metals (As, Ba, Cd, Cr, Cs, Cu, Hg, Mg, Mn, Pb, Se, and Zn) measured in maternal erythrocytes were associated with differentially methylated positions (DMPs) and regions (DMRs) in cord blood and tested if associations persisted in blood collected in mid-childhood. We measured metal concentrations in first-trimester maternal erythrocytes, and DNAm in cord blood (N = 361) and mid-childhood blood (N = 333, 6-10 years) with the Illumina HumanMethylation450 BeadChip. For each metal individually, we tested for DMPs using linear models (considered significant at FDR < 0.05), and for DMRs using comb-p (Sidak p < 0.05). Covariates included biologically relevant variables and estimated cell-type composition. We also performed sex-stratified analyses.

Results: Pb was associated with decreased methylation of cg20608990 (CASP8) (FDR = 0.04), and Mn was associated with increased methylation of cg02042823 (A2BP1) in cord blood (FDR = 9.73 × 10^-6). Both associations remained significant but attenuated in blood DNAm collected at mid-childhood (p < 0.01). Two and nine Mn-associated DMPs were identified in male and female infants, respectively (FDR < 0.05), with two and six persisting in mid-childhood (p < 0.05). All metals except Ba and Pb were associated with ≥ 1 DMR among all infants (Sidak p < 0.05). Overlapping DMRs annotated to genes in the human leukocyte antigen (HLA) region were identified for Cr, Cs, Cu, Hg, Mg, and Mn.

Conclusions: Prenatal metal exposure is associated with DNAm, including DMRs annotated to genes involved in neurodevelopment. Future research is needed to determine if DNAm partially explains the relationship between prenatal metal exposures and health outcomes.

Keywords: DNA methylation; EWAS; Manganese; Metals; Prenatal exposure.

Background: We investigated the apoptotic effects of curcumin in the colon carcinoma cell line SW480.

Methods and results: Cells were treated with 40-200 μM curcumin for 24, 48, and 72 h, and the IC₅₀ values were determined for each time interval. BrdU, caspase-3, and TUNEL staining results and the gene expression of FADD, CASP8, and CASP3 were evaluated. Curcumin treatments significantly inhibited cell proliferation and significantly induced apoptosis for 24, 48, and 72 h. The proportion of BrdU-stained cells in the control groups were 58%, 57% and 61% and 28%, 27%, and 30% in the curcumin treatment groups at 24, 48, and 72 h, respectively. The proportion of apoptotic cells was 28%, 29%, and 28% in the control groups and 59%, 61%, and 60% in the curcumin treatment groups at 24, 48, and 72 h, respectively. As expected, caspase-3 staining also revealed a higher number of apoptotic cells in curcumin treatment groups at 24, 48, and 72 h compared to controls. The proportion of Caspase-3-stained cells in the control groups were 23%, 25%, and 24% and 59%, 60%, and 62% in the curcumin treatment groups at 24, 48, and 72 h, respectively. To prove caspase-3 staining results, FADD, CASP8, and CASP3 gene expressions were evaluated by real-time qPCR. Unlike the immunohistochemical results, no statistically significant upregulation was found at 24 and 48 h, while relative gene expressions of FADD, CASP8, and CASP3 was significantly upregulated at 72 h. The expression level increase was 0.88-, 1.19-, and 2.11-fold for FADD, 1.25-, 1.29-, and 1.59-fold for CASP8, and 1.33-, 1.46-, and 3.00-fold for CASP3 at 24, 48, and 72 h, respectively.

Conclusions: These results suggest that curcumin may be a potential protective or treatment agent against colon cancer; however, further studies on curcumin-rich diets and curcumin bioavailability are required.

Keywords: Apoptosis; Caspase 3; Caspase 8; Curcumin; SW480.

Background/aim: Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults. The aim of this study was to elucidate the molecular pathogenesis of sporadic RCC in Taiwan.

Materials and methods: Fifteen patients with RCC were screened for mutations in the von Hippel-Lindau (VHL) gene by PCR and Sanger sequencing. The methylation status of promoters of 24 tumor suppressor genes by methylation sensitive multiplex ligation-dependent probe amplification analysis was also determined.

Results: Inactivation of the VHL gene was observed in 5 cases: three missense somatic mutations, one promoter methylation, and one small deletion. In RCCs, methylation was most frequently observed in APC (100%), CDKN2B (92.9%), CASP8, MLH1_167, and KLLN (85.7.4%), but not in FHIT, MLH1_463, DAPK1, or HIC1 (0%).

Conclusion: In addition to VHL inactivation, promoter methylation of APC may be a universal pathognomonic event in the tumorigenesis of RCC and a candidate diagnostic and therapeutic biomarker.

Keywords: Renal cell carcinoma; methylation sensitive multiplex ligation-dependent probe amplification analysis; promoter methylation; tumor suppressor gene; von Hippel-Lindau gene.

Purpose: Reovirus propagates with high efficiency in KRAS mutated colorectal cancer (CRC). About 45-50% of CRC patients possess a KRAS mutation. Oncolytic reovirus treatment in combination with chemotherapy was tested in patients possessing KRAS mutated metastatic CRC. This study evaluates the biological responses to reovirus treatment by determining the gene expression patterns in RAS-related signaling pathways.

Methods: Reovirus was administered as a 60-min intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID₅₀) of 3×10¹⁰. Peripheral blood mononuclear cells (PBMCs) were isolated from whole-blood pre- and post-reovirus administration at 48 hr, day-8, and day-15. Clariom_D_Human_Assay was used to determine the expression of vital genes compared to pre-reovirus treatment by RNA sequencing. Using exported sample signals, ΔΔCt method was used to analyze the fold changes of genes within seven gene pathways. Significance was calculated by students-two-tail-t-test. Hierarchical clustering dendrogram was constructed by calculating Pearson's correlation coefficients.

Results: As compared to the control, SOS1[48 hr; 2.49X], RRAS [48 hr; 2.24X], PIK3CB [D8, D15; 2.27X, 3.16X], MIR 16-2 [D15; 1.70X], CHORDC1 [48 hr, D15; 1.89X, 4.54X], RTN4 [48 hr; 4.66X], FAM96A [48 hr; 4.54X], NFKB [D8, D15; 19.0X, 1.42X], CASP8 [D8, D15; 2.11X, 1.77X], and CASP9 [D8; 1.45X] are upregulated post-reovirus. NOS3 [D15; 0.61X], SYNE1 [D8, D15; 0.78X, 0.71X], ANGPT1 [D8; 0.62X], VEGFB [48 hr, D8, D15; 0.44X, 0.28X, 0.28X], JUN [D15; 0.69X], and IGF2 [D8; 0.73X] are downregulated post-reovirus. Fold change values were significant [p<0.05].

Conclusion: This study highlights reovirus as a novel treatment option for KRAS mutated CRC and showcases its effect on the expression of crucial genes.

Keywords: CASP8; CHORDC1; CRC; KRAS; RTN4; VEGFB; reovirus; transcriptome.

Background and Objectives: The effects of Ocimum tenuiflorum essential oil (OTEO) against gastric cancer remain unknown and merit investigation. Materials and Methods: In the present study, the anti-cancer activity of OTEO was examined in a human gastric cancer cell line (AGS). After OTEO treatment, AGS cell viability was determined by an MTT assay, and inhibition of metastasis was determined by cell migration and invasion assays. The expression of apoptosis-related genes in treated AGS cells was determined by qRT-PCR. Results: OTEO significantly decreased AGS cell viability in a dose-dependent manner (IC₅₀ 163.42 µg/mL) and effectively inhibited cell migration and invasion. Morphological examination demonstrated that OTEO induced cell shrinkage, chromatin condensation, and fragmentation, which are considered typical morphologies of apoptotic cell death. Pro-apoptotic genes (TP53, BAX, and BAK) were significantly up-regulated, while anti-apoptotic genes (BCL-2 and BCL-xL) were significantly down-regulated after treatment with OTEO. In addition, significantly increased gene expression was detected for CASP8, CASP9, and CASP3 in AGS cells exposed to OTEO. GC-MS analysis demonstrated that the major compound of OTEO was caryophyllene (25.85%) and α-pinene (11.66%). Conclusions: This in vitro study demonstrates for the first time that OTEO has potential anti-gastric cancer activity and may induce apoptosis in AGS cells through extrinsic and intrinsic pathways.

Keywords: Ocimum tenuiflorum; apoptosis; cell invasion; cell migration; essential oil; gastric cancer.

(1) Background: Over the last decade, genetic counseling clinics have moved from single-gene sequencing to multigene panel sequencing. Multiple genes related to a moderate risk of breast cancer (BC) have emerged, although many questions remain regarding the risks and clinical features associated with these genes. (2) Methods: Ninety-six BC index cases (ICs) with high-risk features for hereditary breast and ovarian cancer (HBOC) and with a previous uninformative result for BRCA1/2 were tested with a panel of 41 genes associated with BC risk. The frequency of pathogenic variants (PVs) was related to the clinical characteristics of BC. (3) Results: We detected a PV rate of 13.5% (excluding two cases each of BRCA1 and MUTYH). Among the 95 assessed cases, 17 PVs were identified in 16 ICs, as follows: BRCA1 (n = 2), CHEK2 (n = 3), ATM (n = 5), MUTYH (n = 2), TP53 (n = 2), BRIP1 (n = 1), CASP8 (n = 1), and MSH2 (n = 1). We also identified a novel loss-of-function variant in CASP8, a candidate gene for increased BC risk. There was no evidence that the clinical characteristics of BC might be related to a higher chance of identifying a PV. (4) Conclusions: In our cohort, which was enriched with families with a high number of BC cases, a high proportion of mutations in ATM and CHEK2 were identified. The clinical characteristics of BC associated with moderate-risk genes were different from those related to BRCA1/2 genes.

Keywords: BRCA1 or BRCA2 negative; germline testing; hereditary breast and ovarian cancer; moderate penetrance genes; next-generation sequencing.

Objective: Fibroblast-like synoviocytes (FLS) play a pivotal role in rheumatoid arthritis (RA) by contributing to synovial inflammation and progressive joint damage. An imprinted epigenetic state is associated with the FLS aggressive phenotype. We identified CASP8 (encoding for caspase-8) as a differentially marked gene and evaluated its pathogenic role in RA FLSs.

Methods: RA FLS lines were obtained from synovial tissues at arthroplasty and used at passage 5-8. Caspase-8 was silenced using small interfering RNA, and its effect was determined in cell adhesion, migration and invasion assays. Quantitative reverse transcription PCR and western blot were used to assess gene and protein expression, respectively. A caspase-8 selective inhibitor was used determine the role of enzymatic activity on FLS migration and invasion. Caspase-8 isoform transcripts and epigenetic marks in FLSs were analyzed in FLS public databases. Crystal structures of caspase-8B and G were determined.

Results: Caspase-8 deficiency in RA FLSs reduced cell adhesion, migration, and invasion independent of its catalytic activity. Epigenetic and transcriptomic analyses of RA FLSs revealed that a specific caspase-8 isoform, variant G, is the dominant isoform expressed (~80% of total caspase-8) and induced by PDGF. The crystal structures of caspase-8 variant G and B were identical except for a unique unstructured 59 amino acid N-terminal domain in variant G. Selective knockdown of caspase-8G was solely responsible for the effects of caspase-8 on calpain activity and cell invasion in FLS.

Conclusion: Blocking caspase-8 variant G could decrease cell invasion in diseases like RA without the potential deleterious effects of nonspecific caspase-8 inhibition.

Understanding the genomic alterations in oral carcinogenesis remains crucial for the appropriate diagnosis and treatment of oral squamous cell carcinoma (OSCC). To unveil the mutational spectrum, in this study, we conducted whole-exome sequencing (WES), using six mutation calling pipelines and multiple filtering criteria applied to 50 paired OSCC samples. The tumor mutation burden extracted from the data set of somatic variations was significantly associated with age, tumor staging, and survival. Several genes (MUC16, MUC19, KMT2D, TTN, HERC2) with a high frequency of false positive mutations were identified. Moreover, known (TP53, FAT1, EPHA2, NOTCH1, CASP8, and PIK3CA) and novel (HYDIN, ALPK3, ASXL1, USP9X, SKOR2, CPLANE1, STARD9, and NSD2) genes have been found to be significantly and frequently mutated in OSCC. Further analysis of gene alteration status with clinical parameters revealed that canonical pathways, including clathrin-mediated endocytotic signaling, NFκB signaling, PEDF signaling, and calcium signaling were associated with OSCC prognosis. Defining a catalog of targetable genomic alterations showed that 58% of the tumors carried at least one aberrant event that may potentially be targeted by approved therapeutic agents. We found molecular OSCC subgroups which were correlated with etiology and prognosis while defining the landscape of major altered events in the coding regions of OSCC genomes. These findings provide information that will be helpful in the design of clinical trials on targeted therapies and in the stratification of patients with OSCC according to therapeutic efficacy.

Keywords: mutation burden; oral cancer; somatic mutation; survival; whole-exome sequencing.

Pyroptosis is a novel kind of cellular necrosis and shown to be involved in cancer progression. However, the diverse expression, prognosis and associations with immune status of pyroptosis-related genes in Hepatocellular carcinoma (HCC) have yet to be analyzed. Herein, the expression profiles and corresponding clinical characteristics of HCC samples were collected from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Then a pyroptosis-related gene signature was built by applying the least absolute shrinkage and selection operator (LASSO) Cox regression model from the TCGA cohort, while the GEO datasets were applied for verification. Twenty-four pyroptosis-related genes were found to be differentially expressed between HCC and normal samples. A five pyroptosis-related gene signature (GSDME, CASP8, SCAF11, NOD2, CASP6) was constructed according to LASSO Cox regression model. Patients in the low-risk group had better survival rates than those in the high-risk group. The risk score was proved to be an independent prognostic factor for overall survival (OS). The risk score correlated with immune infiltrations and immunotherapy responses. GSEA indicated that endocytosis, ubiquitin mediated proteolysis and regulation of autophagy were enriched in the high-risk group, while drug metabolism cytochrome P450 and tryptophan metabolism were enriched in the low-risk group. In conclusion, our pyroptosis-related gene signature can be used for survival prediction and may also predict the response of immunotherapy.

Keywords: hepatocellular carcinoma; immune checkpoint inhibitors; immune infiltrates; prognosis; pyroptosis.

Intestinal homeostasis and the maintenance of the intestinal epithelial barrier are essential components of host defense during gastrointestinal Salmonella Typhimurium infection. Both require a strict regulation of cell death. However, the molecular pathways regulating epithelial cell death have not been completely understood. Here, we elucidated the contribution of central mechanisms of regulated cell death and upstream regulatory components during gastrointestinal infection. Mice lacking Caspase-8 in the intestinal epithelium are highly sensitive towards bacterial induced enteritis and intestinal inflammation, resulting in an enhanced lethality of these mice. This phenotype was associated with an increased STAT1 activation during Salmonella infection. Cell death, barrier breakdown and systemic infection were abrogated by an additional deletion of STAT1 in Casp8^ΔIEC mice. In the absence of epithelial STAT1, loss of epithelial cells was abolished which was accompanied by a reduced Caspase-8 activation. Mechanistically, we demonstrate that epithelial STAT1 acts upstream of Caspase-8-dependent as well as -independent cell death and thus might play a major role at the crossroad of several central cell death pathways in the intestinal epithelium. In summary, we uncovered that transcriptional control of STAT1 is an essential host response mechanism that is required for the maintenance of intestinal barrier function and host survival.

Background: Oral lichen planus confers a 1% risk of transformation to oral squamous cell carcinoma. While prior exome sequencing studies have identified multiple genetic mutations in oral squamous cell carcinoma, mutational analyses of lichen planus-derived OSCC are lacking. We sought to clarify genomic events associated with oral lichen planus transformation.

Methods: Using rigorous diagnostic criteria, we retrospectively identified patients with non-transforming oral lichen planus (i.e. known to be non-transforming with 5 years of clinical follow-up; n=17), transforming oral lichen planus (tissue marginal to oral squamous cell carcinoma, n=9), or oral squamous cell carcinoma arising in lichen planus (n=17). Gene mutational profiles derived from whole-exome sequencing on fixed mucosal specimens were compared amongst the groups.

Results: The four most frequently mutated genes in transforming oral lichen planus and oral squamous cell carcinoma (TP53, CELSR1, CASP8 and KMT2D) identified 12/17 (71%) of oral squamous cell carcinomas and 5/9 (56%) of transforming oral lichen planus but were absent in non-transforming oral lichen planus. These findings suggest alterations in DNA damage response and apoptosis pathways underlie lichen planus-related oral squamous cell carcinoma transformation and are supported by mutational signatures indicative of DNA damage. We identified other known oral squamous cell carcinoma mutations (TRRAP, OBSCN, LRP2) but also previously unreported mutations (TENM3 and ASH1L) in lichen planus-associated oral squamous cell carcinomas.

Conclusions: This study characterized patterns of mutational events present in oral lichen planus associated with squamous cell carcinoma, and in squamous cell carcinoma associated with oral lichen planus, but not in non-transforming oral lichen planus.

Keywords: cancer genetics; carcinogenesis; genomics; lichen planus; oral squamous cell carcinoma.

While studies on embryonic stem cells have been actively conducted, little is known about the epigenetic mechanisms in human embryonic stem cells (hESCs) in extended culture systems. Here, we investigated whether CpG island (CGI) methylation patterns of 24 tumor suppressor genes could be maintained during extended hESC cultures. In total, 10 hESC lines were analyzed. For each cell line, genomic DNA was extracted from early and late passages of cell cultures. CGI methylation levels of 24 tumor suppressor genes were analyzed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), pyrosequencing, and real-time polymerase chain reaction (PCR). Different CGI methylation patterns of CASP8, FHIT, and CHFR genes were identified in between early and late passages in some hESC lines. CGI methylation levels of CASP8 significantly increased at late passage in CHA-36, CHA-40, and CHA-42 cell lines compared to those at early passage. The CGI methylation of the FHIT gene was higher at late passage than at early passage in CHA-15, CHA-31, CHA-32, and iPS (FS)-1 cell lines but decreased at the late passage in CHA-20 and H1 cell lines. Different CGI methylation patterns were detected for the CHFR gene only in iPS (FS)-1, and the level significantly increased at late passage. Thus, our findings show that CGI methylation patterns could be altered during prolonged ESC cultures and examining these epigenetic changes is important to assess the maintenance, differentiation, and clinical usage of stem cells.

The widespread use of chlorothalonil (CTL) has caused environmental residues and food contamination. Although the intestinal epithelial barrier (IEB) is directly involved in the metabolism and transportation of various exogenous compounds, there are few studies on the toxic effects of these compounds on the structure and function of IEB. The disassembly of tight junction (TJ) is a major cause of intestinal barrier dysfunction under exogenous compounds intake, but the precise mechanisms are not well understood. Here, we used Caco-2 cell monolayers as an in vitro model of human IEB to evaluate the toxicity of CTL exposure on the structure and function of IEB. Results showed that CTL exposure increased the paracellular permeability of the monolayers and downregulated messenger RNA levels of the TJ genes (ZO-1, OCLN, and CLDN1), polarity marker gene (SI), and anti-apoptosis gene (BCL-2) but upregulated the messenger RNA levels of apoptosis-related genes, including BAD, BAX, CASP3, and CASP8. Western blot analysis and immunofluorescence assay results showed the decreased levels and disrupted distribution of TJ protein network, including ZO-1 and CLDN1 in CTL-exposed IEB. In addition, the accumulation of intracellular reactive oxygen species, decreased mitochondrial membrane potential, and increased active CASP3 expression were observed in treated IEB. The result of TUNEL assay further confirmed the occurrence of cell apoptosis after CTL exposure. In addition, the phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38, was increased in CTL-exposed IEB. In summary, our results demonstrated that CTL exposure induced IEB dysfunction in Caco-2 cell monolayers by activating the mitogen-activated protein kinase pathway.

Keywords: MAPK; chlorothalonil; intestinal epithelial barrier; permeability; tight junction.

Rewiring of host cytokine networks is a key feature of inflammatory bowel diseases (IBD) such as Crohn's disease (CD). Th1-type cytokines-IFN-γ and TNF-α-occupy critical nodes within these networks and both are associated with disruption of gut epithelial barrier function. This may be due to their ability to synergistically trigger the death of intestinal epithelial cells (IECs) via largely unknown mechanisms. In this study, through unbiased kinome RNAi and drug repurposing screens we identified JAK1/2 kinases as the principal and nonredundant drivers of the synergistic killing of human IECs by IFN-γ/TNF-α. Sensitivity to IFN-γ/TNF-α-mediated synergistic IEC death was retained in primary patient-derived intestinal organoids. Dependence on JAK1/2 was confirmed using genetic loss-of-function studies and JAK inhibitors (JAKinibs). Despite the presence of biochemical features consistent with canonical TNFR1-mediated apoptosis and necroptosis, IFN-γ/TNF-α-induced IEC death was independent of RIPK1/3, ZBP1, MLKL or caspase activity. Instead, it involved sustained activation of JAK1/2-STAT1 signalling, which required a nonenzymatic scaffold function of caspase-8 (CASP8). Further modelling in gut mucosal biopsies revealed an intercorrelated induction of the lethal CASP8-JAK1/2-STAT1 module during ex vivo stimulation of T cells. Functional studies in CD-derived organoids using inhibitors of apoptosis, necroptosis and JAKinibs confirmed the causative role of JAK1/2-STAT1 in cytokine-induced death of primary IECs. Collectively, we demonstrate that TNF-α synergises with IFN-γ to kill IECs via the CASP8-JAK1/2-STAT1 module independently of canonical TNFR1 and cell death signalling. This non-canonical cell death pathway may underpin immunopathology driven by IFN-γ/TNF-α in diverse autoinflammatory diseases such as IBD, and its inhibition may contribute to the therapeutic efficacy of anti-TNFs and JAKinibs.

Introduction: The somatic mutational profile of oral squamous cell carcinoma (OSCC) among Japanese patients has been less investigated, partly because of the rarity of the tumor. Moreover, previous studies have either used formalin-fixed paraffin-embedded samples or lacked paired normal tissues. We aimed to determine somatic mutations in the exomes of 76 genes, including 50 driver genes of solid cancers and NOTCH-related genes, some of which are previously reported as frequently mutated in head and neck squamous cell carcinoma or OSCC.

Materials and methods: We used fresh-frozen tumor/normal-paired samples from 98 treatment-naïve Japanese patients with OSCC and analyzed their correlations with clinicopathological characteristics and survival.

Results: We identified 136 exonic mutations, including 78 non-synonymous mutations, 13 synonymous mutations, 22 nonsense mutations, 2 non-frameshift deletions, 11 frameshift deletion, and 5 each of splice-site and frameshift insertions. The most frequently mutated genes were TP53 (36.7%), FAT1 (9.2%), NOTCH1 (8.2%), CDKN2A (7.1%), ZFHX4 (5.1%), CASP8 (4.1%), EP300 (4.1%), and KMT2D (4.1%). We followed up 90 of the 98 patients for 3 years. Among them, TP53 mutation was associated with significantly shorter 3-year disease-free survival. Most of the identified TP53 mutations occurred in the DNA-binding domain and were functionally deleterious.

Discussion: Our findings and the mutation spectra can contribute to the development of a therapeutic strategy for Japanese patients with OSCC.

Keywords: Clinical oncology; Head and neck cancer; Japanese patients; OSCC; Oral squamous cell carcinoma; Somatic mutation.

Objective: Pyroptosis represents an emerging inflammatory form of programmed cell death. Herein, specific functions and clinical implications of pyroptosis-related genes were systematically characterized in breast cancer. Methods: Expression, somatic mutation and copy number variation of 33 pyroptosis-related genes were assessed in breast cancer from TCGA dataset. Their interactions, biological functions and prognostic values were then observed. By stepwise Cox regression analysis, a pyroptosis-related gene signature was generated. The predictive efficacy in survival was examined by survival analyses, ROCs, univariate and multivariate analyses and subgroup analyses. Associations between risk score (RS) and cancer immunity cycle, HLA, immune cell infiltrations, and immune checkpoints were analyzed. Results: Most of pyroptosis-related genes were abnormally expressed in breast cancer. CASP8, NLRC4, NLRP3, NLRP2, PLCG1, NLRP1, NLRP7, SCAF11, GSDMC, and NOD1 occurred somatic mutations as well as most of them had high frequency of CNV. There were closely interactions between them. These genes were distinctly enriched in immune-related processes. A three-gene signature was generated, containing IL-18, GSDMC, and TIRAP. High RS predicted poorer overall survival, progression, and recurrence. After verification, this RS was an independent and sensitive predictive index. This RS was negatively correlated to cancer immunity cycle. Also, low RS was characterized by high HLA, immune cell infiltrations and immune checkpoints. A nomogram including age and RS was generated for accurately predicting 5-, 8-, and 10-year survival probabilities. Conclusion: Pyroptosis-related genes exert key roles in cancer immunity and might be applied as a prognostic factor of breast cancer.

Keywords: breast cancer; immune; nomogram; prognosis; pyroptosis; signature.

Ozone (O₃) is the predominant oxidant air pollutant associated with airway inflammation, lung dysfunction, and the worsening of preexisting respiratory diseases. We previously demonstrated the injurious roles of pulmonary immune receptors, tumor necrosis factor receptor (TNFR), and toll-like receptor 4, as well as a transcription factor NF-κB, in response to O₃ in mice. In the current study, we profiled time-dependent and TNFR- and NF-κB-regulated lung transcriptome changes by subacute O₃ to illuminate the underlying molecular events and downstream targets. Mice lacking Tnfr1/Tnfr2 (Tnfr^-/-) or Nfkb1 (Nfkb1^-/-) were exposed to air or O₃. Lung RNAs were prepared for cDNA microarray analyses, and downstream and upstream mechanisms were predicted by pathway analyses of the enriched genes. O₃ significantly altered the genes involved in inflammation and redox (24 h), cholesterol biosynthesis and vaso-occlusion (48 h), and cell cycle and DNA repair (48-72 h). Transforming growth factor-β1 was a predicted upstream regulator. Lack of Tnfr suppressed the immune cell proliferation and lipid-related processes and heightened epithelial cell integrity, and Nfkb1 deficiency markedly suppressed lung cell cycle progress during O₃ exposure. Common differentially regulated genes by TNFR and NF-κB1 (e.g., Casp8, Il6, and Edn1) were predicted to protect the lungs from cell death, connective tissue injury, and inflammation. Il6-deficient mice were susceptible to O₃-induced protein hyperpermeability, indicating its defensive role, while Tnf-deficient mice were resistant to overall lung injury caused by O₃. The results elucidated transcriptome dynamics and provided new insights into the molecular mechanisms regulated by TNFR and NF-κB1 in pulmonary subacute O₃ pathogenesis.

Keywords: IL-6; NF-κB; TNF receptor; lung; mice; microarray; ozone.