SARS-CoV-2 infection has a risk to develop into life-threatening COVID-19 disease. Whereas age, hypertension, and chronic inflammatory conditions are risk factors, underlying host factors and markers for disease severity, e.g. requiring intensive care unit (ICU) treatment, remain poorly defined. To this end, we longitudinally profiled blood inflammation markers, antibodies, and 101 plasma proteins of hospitalized COVID-19 patients who did or did not require ICU admission. While essentially all patients displayed SARS-CoV-2-specific antibodies and virus-neutralization capacity within 12-15 days, a rapid, mostly transient upregulation of selective inflammatory markers including IL-6, CXCL10, CXCL11, IFNγ, IL-10, and monocyte-attracting CCL2, CCL7 and CCL8, was particularly evident in ICU patients. In addition, there was consistent and sustained upregulation of apoptosis-associated proteins CASP8, TNFSF14, HGF, and TGFB1, with HGF discriminating between ICU and non-ICU cohorts. Thus, COVID-19 is associated with a selective inflammatory milieu within which the apoptotic pathway is a cardinal feature with potential to aid risk-based patient stratification.

OBJECTIVE

To investigate, by genotyping CASP8 (-652 6N del/ins) and CASP9 (-1263 A>> G; -293 19N del/ins), whether inactivation of apoptosis by genetic polymorphism of caspases 8 and 9 play an integral role in the mechanism of cancer development. To investigate the role of these polymorphisms in susceptibility to early development of hormone refractory prostate cancer.

METHODS

The study included 175 histologically confirmed cases of prostate cancer and 198 age and ethnicity matched healthy controls. CASP9-1263 A>> G polymorphism was genotyped using the polymerase chain reaction-restriction fragment length polymorphism method. CASP9-293 del/ins and CASP8-652 del/ins polymorphisms were genotyped and the deletion pattern analysed by polyacrylamide gel electrophoresis.

RESULTS

Our results demonstrated that presence of CASP9-1263 G allele was associated with reduced risk for prostate cancer (odds ratio 0.6, 95%CI 0.39-0.92, P= 0.02). Other variant CASP9 was not associated with prostate cancer risk. Coincidentally, the presence of CASP9-1263 G allele was associated with increased risk for progression of prostate cancer to bone metastasis (odds ratio -2.28, 95%CI 1.14-4.53, P= 0.02). CASP8-652 (+/-) genotype was associated with increased hazard for early development of hormone refractory prostate cancer (hazard ratio 2.44, 95%CI 1.2-5.85, P= 0.045).

CONCLUSIONS

Our results support the hypothesis that variants of CASP9 may influence the susceptibility to prostate cancer and its progression to bone metastasis. CASP8 polymorphism may influence the progression of prostate cancer disease to a hormone refractory state.

COMPASS is a profile-based method for the detection of remote sequence similarity and the prediction of protein structure. Here we describe a recently improved public web server of COMPASS, http://prodata.swmed.edu/compass. The server features three major developments: (i) improved statistical accuracy; (ii) increased speed from parallel implementation; and (iii) new functional features facilitating structure prediction. These features include visualization tools that allow the user to quickly and effectively analyze specific local structural region predictions suggested by COMPASS alignments. As an application example, we describe the structural, evolutionary and functional analysis of a protein with unknown function that served as a target in the recent CASP8 (Critical Assessment of Techniques for Protein Structure Prediction round 8). URL: http://prodata.swmed.edu/compass.

Acute promyelocytic leukemia (APL) is one of the most life-threatening hematological malignancies. Defects in the cell growth and apoptotic pathways are responsible for both disease pathogenesis and treatment resistance. Therefore, pro-apoptotic agents are potential candidates for APL treatment. Kaempferol is a flavonoid with antioxidant and anti-tumor properties. This study was designed to investigate the cytotoxic, pro-apoptotic, and differentiation-inducing effects of kaempferol on HL-60 and NB4 leukemia cells. Resazurin assay was used to determine cell viability following treatment with kaempferol (12.5-100 μM) and all-trans retinoic acid (ATRA; 10 μM; used as a positive control). Apoptosis and differentiation were also detected using propidium iodide and NBT staining techniques, respectively. Furthermore, the expression levels of genes involved in apoptosis (PI3 K, AKT, BCL2, BAX, p53, p21, PTEN, CASP3, CASP8, and CASP9), differentiation (PML-RAR and HDAC1), and multi-drug resistance (ABCB1 and ABCC1) were determined using quantitative real-time PCR. The protein expressions of Bax/Bcl2 and casp3 were confirmed using Western blot. The results showed that kaempferol decreased cell viability and increased subG1 population in the tested leukemic cells. This effect was associated with decreased expression of Akt, BCL2, ABCB1, and ABCC1 genes, while the expression of CASP3 and BAX/BCL-2 ratio were significantly increased at both gene and protein levels. Kaempferol promoted apoptosis and inhibited multidrug resistance in a concentration-dependent manner, without any differential effect on leukemic cells. In conclusion, this study suggested that kaempferol may be utilized as an appropriate alternative for ATRA in APL patients.

BACKGROUND

MOBI is a web server for the identification of structurally mobile regions in NMR protein ensembles. It provides a binary mobility definition that is analogous to the commonly used definition of intrinsic disorder in X-ray crystallographic structures. At least three different use cases can be envisaged: (i) visualization of NMR mobility for structural analysis; (ii) definition of regions for reliable comparative modelling in protein structure prediction and (iii) definition of mobility in analogy to intrinsic disorder. MOBI uses structural superposition and local conformational differences to derive a robust binary mobility definition that is in excellent agreement with the manually curated definition used in the CASP8 experiment for intrinsic disorder in NMR structure. The output includes mobility-coloured PDB files, mobility plots and a FASTA formatted sequence file summarizing the mobility results.

BACKGROUND

The MOBI server and supplementary methods are available for non-commercial use at URL: http://protein.bio.unipd.it/mobi/.

Protein template identification is essential to protein structure and function predictions. However, conventional whole-chain threading approaches often fail to recognize conserved substructure motifs when the target and templates do not share the same fold. We developed a new approach, SEGMER, for identifying protein substructure similarities by segmental threading. The target sequence is split into segments of two to four consecutive or nonconsecutive secondary structural elements, which are then threaded through PDB to identify appropriate substructure motifs. SEGMER is tested on 144 nonredundant hard proteins. When combined with whole-chain threading, the TM-score of alignments and accuracy of spatial restraints of SEGMER increase by 16% and 25%, respectively, compared with that by the whole-chain threading methods only. When tested on 12 free modeling targets from CASP8, SEGMER increases the TM-score and contact accuracy by 28% and 48%, respectively. This significant improvement should have important impact on protein structure modeling and functional inference.

Caspase (CASP) 3, 8, 9 are important caspases in the apoptosis pathway and play important roles in development and progression of cancer. A case-control study with 451 colorectal cancer (CRC) patients and 631 cancer-free controls were carried out, and CRC patients followed up, to investigate the associations between three main polymorphisms and colorectal cancer risk and prognosis, and their potential interactions with environmental factors on CRC risk among Chinese people. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction-single strand conformation polymorphism sequencing. Odds ratio (OR), hazard ratio (HR) and their 95 % confidence intervals (CIs) were estimated with unconditional logistic-regression and Cox proportion hazard model. Individuals harboring the CASP8 -652 6N ins/del plus del/del genotype had a slightly lower risk for CRC compared those with ins/ins genotype (adjusted OR = 0.77, 95 % CI 0.59-0.99, P = 0.04). Significant associations between CASP3 -928 GG genotype and CASP9 -1263 GG genotype and reduced risk of rectal cancer were observed (adjusted OR = 0.56, 95 % CI 0.34-0.92, P = 0.02; adjusted OR = 0.59, 95 % CI 0.36-0.95, P = 0.03, respectively). There was a marginal significant association between CASP8 -652 6N ins/del polymorphism and CRC prognosis (ins/del versus ins/ins, adjusted HR = 0.69, 95 % CI 0.48-0.99, P = 0.04). These findings suggested these polymorphisms and their combinations with dietary factors may be associated with the development of CRC. CASP8 -652 6N ins/del polymorphism may be an independent survival predictor for CRC.

OBJECTIVE

Comparison of subjects with extreme phenotypes of cancer susceptibility and tolerance allows to detect low-penetrance gene-disease interactions with a relatively small study size.

METHODS

We analyzed the distribution of 19 coding apoptotic gene polymorphisms (Bid Gly10Ser; Casp2 Leu141Val; Casp5 Ala90Thr and Val318Leu; Casp7 Glu255Asp; Casp8 His302Asp; Casp9 Val28Ala, His173Arg and Arg221Gln; Casp10 Ile479Leu; Faim Thr117Ala and Ser127Leu; DR4 Arg141His, Thr209Arg, Ala228Glu and Lys441Arg; Survivin Lys129Glu; TNFR1 Gln121Arg; XIAP Pro423Gln) in 121 breast cancer patients with clinical features of a hereditary predisposition (family history and/or early onset and/or bilaterality) and 142 elderly tumor-free women.

RESULTS

None of the individual single nucleotide polymorphisms (SNPs) demonstrated an association with breast cancer risk. The analysis of gene interactions revealed that the combination of XIAP Pro423Gln (rs5956583) AA genotype with Casp7 Glu255Asp (rs2227310) CG genotype appeared to prevail in "supercases" relative to "supercontrols" (25/121 [21%] vs 11/142 [8%], P = 0.002). We attempted to validate this association in the second round of case-control analysis, which involved 519 randomly selected breast cancer patients and 509 age-matched healthy women, but no difference was detected upon this comparison.

CONCLUSIONS

Coding apoptotic gene polymorphisms do not play a major role in BC predisposition. The results of this investigation may be considered while designing future studies on breast cancer-associated candidate SNPs.

There is an increasing incidence of esophageal adenocarcinoma (EA) among younger people in the western populations. However, the association between genetic polymorphisms and the age of EA onset is unclear. In this study, 1330 functional/tagging single-nucleotide polymorphisms (SNPs) from 354 cancer-related genes were genotyped in 335 white EA patients. Twenty important SNPs that have the highest importance scores and lowest classification error rate were identified by the random forest algorithm to be associated with early onset of EA (age ≤ 55 years). Subsequent logistic regression analysis indicated that 10 SNPs (rs2070744 of NOS3, rs720321 of BCL2, rs17757541 of BCL2, rs11775256 of TNFRSF10A, rs1035142 of CASP8, rs2236302 of MMP14, rs4740363 of ABL1, rs696217 of GHRL, rs2445762 of CYP19A1, and rs11941492 of VEGFR2/KDR) were significantly associated with early onset of EA (≤55 vs >55 years, all P < .05 after adjusting for co-variates and false discovery rate). Among them, five SNPs in the NOS3, BCL2, TNFRSF10A, and CASP8 genes were known to be involved in apoptosis processes. In Kaplan-Meier analyses, rs2070744 of NOS3, rs720321 of BCL2, and rs1035142 of CASP8 were also significantly associated with early onset of EA. Moreover, there was a higher risk of developing EA at a younger age when one had more risk genotypes. In conclusion, polymorphisms in cancer-related genes, especially those in the apoptotic pathway, play an important role in the development of younger-aged EA in a dose-response manner.

BACKGROUND

In a previous microarray expression analysis, the authors identified candidate genes that were expressed differentially between ovarian tumors with low malignant potential and invasive serous epithelial ovarian tumors. Among them, the apoptosis-related candidate genes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), caspase 8 (CASP8), FLICE-inhibitory protein (FLIP), and cytochrome C (CYC) were identified.

METHODS

For the current study, the authors conducted immunohistochemical analyses of a tissue array comprised of 235 serous tumors of different grades and stages to evaluate whether there was differential protein expression for these candidates and for the 4 death cell receptors of Trail: Dr4, Dr5, DcR1, and DcR2.

RESULTS

All proteins except DcR1 and DcR2 had significantly differential expression levels between grade 0 tumors (low malignant potential) and grade 2 and 3 tumors. Trail also showed differential expression between grade 0 tumors and grade 1 tumors. When all tumors were compared, the expression levels of Trail, Dr4, Dr5, DcR1, and Flip differed significantly between early-stage and advanced-stage disease. High Dr5 expression was associated with a poor prognosis in patients who had invasive tumors and in the subgroup of patients who had grade 3 tumors. Furthermore, the combinations of 2 proteins (Trail and Dr5, DcR2 and Cyc, Flip and Dr5, Flip and DcR2, DcR1 and Dr5 or Dr4 and Flip) revealed an association with patient prognosis.

CONCLUSIONS

The identification of new proteins in the initial diagnosis and prognosis of patients with epithelial ovarian cancer may lead to a better understanding of the disease, highlighting new potential therapeutic targets, and may be useful in patient management.

BACKGROUND

We propose a novel method for scoring the accuracy of protein binding site predictions-the Binding-site Distance Test (BDT) score. Recently, the Matthews Correlation Coefficient (MCC) has been used to evaluate binding site predictions, both by developers of new methods and by the assessors for the community-wide prediction experiment-CASP8. While being a rigorous scoring method, the MCC does not take into account the actual 3D location of the predicted residues from the observed binding site. Thus, an incorrectly predicted site that is nevertheless close to the observed binding site will obtain an identical score to the same number of non-binding residues predicted at random. The MCC is somewhat affected by the subjectivity of determining observed binding residues and the ambiguity of choosing distance cutoffs. By contrast the BDT method produces continuous scores ranging between 0 and 1, relating to the distance between the predicted and observed residues. Residues predicted close to the binding site will score higher than those more distant, providing a better reflection of the true accuracy of predictions. The CASP8 function predictions were evaluated using both the MCC and BDT methods and the scores were compared. The BDT was found to strongly correlate with the MCC scores while also being less susceptible to the subjectivity of defining binding residues. We therefore suggest that this new simple score is a potentially more robust method for future evaluations of protein-ligand binding site predictions.

BACKGROUND

http://www.reading.ac.uk/bioinf/downloads/.

BACKGROUND

Cytoplasmic viral double-stranded RNA (dsRNA) is detected by a class of ubiquitous cytoplasmic RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation antigen-5 (MDA5), which initiate a signaling cascade via their common adaptor called interferon-β (IFN-β) promoter stimulator-1 (IPS-1). This leads to the production of proinflammatory and antiviral cytokines, the type I Interferons, via mainly nuclear factor kappa B (NF-κB) and interferon response factor-3 (IRF3) transcription factors. Fas-associated death domain (FADD) protein, receptor-interacting protein (RIP1), caspase-8 and tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) protein, all traditionally associated with death receptor signaling, are also involved in RIG-I/MDA5 signaling pathway. We previously showed that FLIP (Flice-like inhibitory protein), also designated as cflar (CASP8 and FADD-like apoptosis regulator), negatively regulates lipopolysaccharide (LPS)-induced toll-like receptor 4 (TLR4) signaling in endothelial cells and mouse embryonic fibroblasts (MEFs) and protected against TLR4-mediated apoptosis.

RESULTS

In this study, we investigated the role of FLIP in cellular response to cytoplasmic polyinosinic:polycytidylic acid, poly(I:C), a synthetic analog of dsRNA. Consistent with the previously described role of FADD in RIG-I/MDA5-mediated apoptosis, we found that FLIP-/- MEFs were more susceptible to killing by cytoplasmic poly(I:C). However, FLIP-/- MEFs also exhibited markedly increased expression of NF-κB-and IRF3- dependent genes in response to cytoplasmic poly(I:C). Importantly, reconstitution of FLIP in FLIP-/-MEFs reversed the hyper-activation of IRF3- and NF-κB-mediated gene expression. Further, we found that caspase-8 catalytic activity was not required for cytoplasmic poly(I:C)-mediated NF-κB and IRF3 signaling.

CONCLUSIONS

These results provide evidence for a crucial dual role for FLIP in antiviral responses to cytoplasmic dsRNA: it protects from cytoplasmic dsRNA-mediated cell death while down-regulating IRF3-and NF-κB-mediated gene expression. Since the pathogenesis of several viral infections involves a heightened and dysregulated cytokine response, a possible therapy could involve modulating FLIP levels.

The cytokine tumor necrosis factor alpha (TNF-α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF-κB) activation, apoptosis signaling by way of caspase-8 (Casp8), and activation of cJun N-terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8(Δhepa) ) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8(Δhepa) mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF-dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor-interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF-κB and JNK/cJun related signals. In order to define the role of NF-κB in this setting we blocked NF-κB activation in Casp8(Δhepa) mice by concomitant inactivation of the NF-κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8(Δhepa) mice but resulted in incomplete termination of the regeneration process and hepatomegaly.

CONCLUSIONS

Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF-κB, and JNK activation. While loss of Casp8 triggers NF-κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly.

Supratentorial primitive neuroectodermal tumors (sPNET) and atypical teratoid/rhabdoid tumors (AT/RT) of the CNS represent a biological and clinical enigma, despite advances in both molecular techniques and clinical management for these two rare embryonal brain tumors of childhood. Epigenetic changes hold great potential as possible disease mechanisms and may be manipulated therapeutically. We thus studied aberrant methylation of the genes RASSF1A and CASP8 and its consequence on expression in cell lines and primary tumors using a combination of semiquantitative methylation specific PCR (MSP), bisulfite sequencing and RT-PCR. In all, 17 samples of autopsy-derived normal appearing brain served as controls. Opposed to control tissues 19/24 sPNET and 4/6 AT/RT demonstrated aberrant methylation for the RASSF1A promoter region. Treatment of cell lines using 5-Aza-2'-deoxycytidine (5AZA) alone or in combination with trichostatin A (TSA) succeeded in re-establishing expression of RASSF1A in cell lines derived from a renal rhabdoid, an AT/RT and a medulloblastoma. A 5' CpG-rich region of CASP8 was methylated in normal tissues and in tumors. However, CASP8 showed inconsistent expression patterns in normal and tumor tissues. Our results indicate that aberrant methylation of the RASSF1A promoter region may be of importance in the origin and progression of sPNET and AT/RT while the analysed 5'-CpG rich region of the CASP8 gene does not seem to play an important role in these tumors. Further studies of epigenetic changes in these rare tumors are warranted as their biology remains obscure and treatment efforts have been rather unsuccessfull.

Caspase-8 (CASP8) is a key controller of apoptosis, and its deregulation plays an important role in carcinogenesis. To evaluate the role of CASP8 polymorphisms in gallbladder cancer (GBC), we examined the risk associated with three single-nucleotide polymorphisms (SNPs) in a case-control study in North Indian population. Genotypes and haplotypes of the CASP8 polymorphisms (-652 6N ins/del; rs3834129, Ex13 + 51G>> C; rs1045485 and IVS12-19 G>> A; rs3769818) were determined for 230 GBC patients and 230 cancer-free controls randomly selected from the population. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated in multivariate logistic regression analysis for the association of individual SNPs and haplotypes with GBC. Carriers for the "del" allele of rs3834129 SNP were associated with a 0.60-fold decreased risk for GBC (95% CI = 0.42-0.88; P(trend) = 0.005). In the combined analysis of the three CASP8 variants, we found that the individuals with the diplotypes carrying two copies of the common CASP8 del-G-G haplotype had 0.35-fold reduced risk (95% CI = 0.14-0.85) when compared with the diplotype containing 0-1 copy. The false-positive report probability (FPRP) approach advocated that these results were noteworthy (FPRP < 0.5). The molecular modeling results of rs1045485 polymorphism indicated that the overall configuration of both wild-type and polymorphic CASP8 protein were similar, with negligible deviation at the site of the polymorphism itself. In summary, low penetrance variants in CASP8 gene may alter the susceptibility toward GBC.

BACKGROUND

Caspase8 influences carcinogenesis through regulation of apoptosis, hyperproliferation, and metastasis. Role of genetic variations in caspase8 has been explored in various cancers; however, their predictive and prognostic role in esophageal cancer is poorly understood.

METHODS

We investigated the association of two potential caspase8 polymorphisms: CASP8 -652 6N del and CASP8 IVS12-19 G>A polymorphisms with susceptibility and survival of 259 esophageal squamous cell carcinoma (ESCC) cases and 259 cancer-free controls from northern Indian population using PCR/PCR RFLP method.

RESULTS

CASP8 IVS12-19 AA genotype was found to be associated with significant increased risk of ESCC (odds ratio (OR) 3.28, 95% confidence interval (CI) 1.04-10.29) specifically in male subjects (OR 3.71, 95% CI 1.01-13.35) with lower third tumor anatomical location (OR 6.00, 95% CI 1.60-22.55). Kaplan-Meier and Cox Regression analysis showed lower median survival (7.13 months vs. 25.21 months) and greater hazard of death (HR 3.40, 95% CI 1.38-7.90) with CASP8 IVS12-19 AA genotype in ESCC cases compared to IVS12-19 GG genotype. However, no association of CASP8 -652 6N del polymorphism with susceptibility and prognosis of ESCC was observed.

CONCLUSIONS

CASP8 IVS12-19 G>A but not CASP8 -652 6N del polymorphism may modulate risk of ESCC and its survival outcome in northern Indian population.

OBJECTIVE

This study was conducted to evaluate the potential role of CASP8 genetic polymorphisms in the etiology of breast cancer in a case-control study, Korea.

METHODS

Incident breast cancer cases confirmed histologically (n = 1,599) were recruited from two hospitals in Seoul during 2001-2005. Control subjects (n = 1,536) were selected from the Health Examinee Cohort from Seoul and Gyeonggi Province surrounding Seoul, Korea. Three SNPs (D302H D>> H, 5'-UTR C>> T, and K337K G>> A) were genotyped by the primer extension assay. The CASP8 D302H, which was not polymorphic in 48 samples, was excluded in further genotyping. Odds ratios and 95% confidential intervals (95% CIs) were estimated by unconditional logistic regression model adjusted for age at enrollment, education, age at first full-term pregnancy, cigarette smoking, and family history of breast cancer.

RESULTS

The 5'-UTR T allele containing genotypes (CT/TT) were associated with an increased risk of breast cancer, compared with those with the CC genotype (OR = 1.13, 95% CI = 0.95-1.34; and OR = 1.48, 95% CI = 1.04-2.10, respectively; P-trend = 0.02). When stratified by the estrogen and progesterone receptor status, the association between the 5'-UTR T allele and breast cancer risk was prominent in ER(+) and PR(+) cases among pre-menopausal women (OR = 1.31, 95% CI = 1.00-1.72 and OR = 1.40, 95% CI = 1.06-1.85, respectively), whereas the association was found prominent in ER(-) or PR(-) cases (OR = 1.32, 95% CI = 0.93-1.87 and OR = 1.42, 95% CI = 1.04-1.94, respectively) among post-menopausal women.

CONCLUSIONS

Our results thus suggest that the CASP8 5'-UTR C>> T are associated with breast cancer risks and the effect may be modified by estrogen and progesterone receptor status.

Necroptosis, a caspase-independent, receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1)/RIPK3-dependent necrotic cell death, occurs in cells when apoptosis is blocked. A high level of macroautophagy (herein referred to as autophagy) is usually detected in necroptotic cells, although it is still controversial as to whether excessive autophagy leads to cell death or is cytoprotective. In a recently published paper, we show that the anti-apoptotic protein CFLAR (CASP8 and FADD-like apoptosis regulator) long isoform (CFLARL) plays a critical role in all three fundamental intracellular processes: autophagy, necroptosis, and apoptosis in T lymphocytes. CFLARL-deficient T cells suffer from severe cell death upon T cell receptor stimulation, in which both apoptosis and necroptosis are involved. Autophagy is enhanced in both naïve and activated CFLARL-deficient T cells and plays a cytoprotective function. Here, we summarize our findings and discuss the future direction in the study of the interplay of autophagy, apoptosis and necroptosis in T lymphocytes.

BACKGROUND

Wilms tumours (WTs) are characterised by several hallmarks that suggest epimutations such as aberrant DNA methylation are involved in tumour progression: loss of imprinting at 11p15, lack of recurrent mutations and formation of nephrogenic rests (NRs), which are lesions of retained undifferentiated embryonic tissue that can give rise to WTs.

METHODS

To identify such epimutations, we performed a comprehensive methylome analysis on 20 matched trios of micro-dissected WTs, NRs and surrounding normal kidneys (NKs) using Illumina Infinium HumanMethylation450 Bead Chips and functionally validated findings using RNA sequencing.

RESULTS

Comparison of NRs with NK revealed prominent tissue biomarkers: 629 differentially methylated regions, of which 55% were hypermethylated and enriched for domains that are bivalent in embryonic stem cells and for genes expressed during development (P = 2.49 × 10(-5)). Comparison of WTs with NRs revealed two WT subgroups; group-2 WTs and NRs were epigenetically indistinguishable whereas group-1 WTs showed an increase in methylation variability, hypomethylation of renal development genes, hypermethylation and relative loss of expression of cell adhesion genes and known and potential new WT tumour suppressor genes (CASP8, H19, MIR195, RB1 and TSPAN32) and was strongly associated with bilateral disease (P = 0.032). Comparison of WTs and NRs to embryonic kidney highlighted the significance of polycomb target methylation in Wilms tumourigenesis.

CONCLUSIONS

Methylation levels vary during cancer evolution. We have described biomarkers related to WT evolution from its precursor NRs which may be useful to differentiate between these tissues for patients with bilateral disease.

Toll-like receptor 2 (TLR2) is involved in innate immunity in the brain and in the cascade of events after ischemic stroke. The aim of this study was to get an insight into the expression of genes related to TLR2 signaling pathway and associated with inflammation and apoptosis in the later stages of brain response after ischemic injury. Middle cerebral artery occlusion was performed on both wild-type and TLR2(-/-) mice followed by real-time PCR to measure the relative expression of selected genes. In TLR2(-/-) mice expression of genes involved in proinflammatory response was decreased after cerebral ischemia. Tnf was the most prominent cytokine active in the late phase of recovery. Contrary to proinflammatory genes, the expression of Casp8, as a hallmark of apoptosis, was increased in TLR2(-/-) mice, in particular in the late phase of recovery.

Trenbolone, an anabolic androgen, and flutamide, an antiandrogen, are prototypical model compounds for agonism and antagonism of the androgen receptor. We hypothesized that 48 h exposures of female fathead minnows (Pimephales promelas) to environmentally relevant concentrations of these chemicals would alter genes regulated by the androgen receptor and that a mixture of the two compounds would block the effects. Gene expression in the ovaries was analyzed using a fathead minnow-specific 22,000-gene microarray. Flutamide altered abouttwicethe number of genes astrenbolone, most of which appeared to be through pathways not associated with the androgen receptor. A group of 70 genes, of which we could identify 37, were reciprocally regulated by trenbolone and flutamide. These are candidates for specific biomarkers for androgen receptor mediated gene expression. Four genes stand out as specifically related to reproduction: sperm associated antigen 8 (SPAG8), CASP8 and FADD-like apoptosis regulator (CFLAR), corticotropin releasing hormone (CRH), and 3beta-hydroxysteroid dehydrogenases (3beta-HSD). Three notable transcriptional regulators including myelocytomatosis viral oncogene homologue (MYC), Yin Yang 1 (YY1), and interferon regulator factor 1 (IRF1) may function as early molecular switches to control phenotypic changes in ovary tissue architecture and function in response to androgen or antiandrogen exposure.

BACKGROUND

MetastamiRs have momentous clinical relevance and have been correlated with disease progression in many tumors. In this study, we identified neuroblastoma metastamiRs exploiting unique mouse models of favorable and high-risk metastatic human neuroblastoma. Further, we related their deregulation to the modulation of target proteins and established their association with clinical outcomes.

RESULTS

Whole genome miRNA microarray analysis identified 74 metastamiRs across the manifold of metastatic tumors. RT-qPCR on select miRNAs validated profile expression. Results from bio-informatics across the ingenuity pathway, miRCancer, and literature data-mining endorsed the expression of these miRNAs in multiple tumor systems and showed their role in metastasis, identifying them as metastamiRs. Immunoblotting and TMA-IHC analyses revealed alterations in the expression/phosphorylation of metastamiRs' targets, including ADAMTS-1, AKT1/2/3, ASK1, AURKβ, Birc1, Birc2, Bric5, β-CATENIN, CASP8, CD54, CDK4, CREB, CTGF, CXCR4, CYCLIN-D1, EGFR, ELK1, ESR1, CFOS, FOSB, FRA, GRB10, GSK3β, IL1α, JUND, kRAS, KRTAP1, MCP1, MEGF10, MMP2, MMP3, MMP9, MMP10, MTA2, MYB, cMYC, NF2, NOS3, P21, pP38, PTPN3, CLEAVED PARP, PKC, SDF-1β, SEMA3D, SELE, STAT3, TLR3, TNFα, TNFR1, and VEGF in aggressive cells ex vivo and in a manifold of metastatic tumors in vivo. miRNA mimic (hsa-miR-125b, hsa-miR-27b, hsa-miR-93, hsa-miR-20a) and inhibitor (hsa-miR-1224-3p, hsa-miR-1260) approach for select miRNAs revealed the direct influence of the altered metastamiRs in the regulation of identified protein targets. Clinical outcome association analysis with the validated metastamiRs' targets corresponded strongly with poor overall and relapse-free survival.

CONCLUSIONS

For the first time, these results identified a comprehensive list of neuroblastoma metastamiRs, related their deregulation to altered expression of protein targets, and established their association with poor clinical outcomes. The identified set of distinctive neuroblastoma metastamiRs could serve as potential candidates for diagnostic markers for the switch from favorable to high-risk metastatic disease.