The performance of the protein structure prediction server pro-sp3-TASSER in CASP8 is described. Compared to CASP7, the major improvement in prediction is in the quality of input models to TASSER. These improvements are due to the PRO-SP(3) threading method, the improved quality of contact predictions provided by TASSER_2.0, multiple short TASSER simulations for building the full-length model, and the accuracy of model selection using the TASSER-QA quality assessment method. Finally, we analyze the overall performance and highlight some successful predictions of the pro-sp3-TASSER server.

Idiopathic talipes equinovarus, also known as clubfoot, is a common birth defect occurring in one of 1000 live births. It is a complex disorder in which multiple genes and environmental factors may play an etiologic role. Several chromosomal deletion regions, including 2q31-33, are associated with talipes equinovarus and may harbor genes that contribute to the idiopathic talipes equinovarus phenotype. Previously, two STRs in the 2q31-33, GATA149B10 and D2S1371, showed linkage with association to idiopathic talipes equinovarus. Single nucleotide polymorphisms (SNPs) in three apoptotic genes (Casp8, Casp10, and CFLAR) near GATA149B10 were genotyped in idiopathic talipes equinovarus families. rs3731714 in Casp10 showed linkage with association, suggesting variation in the apoptotic gene pathway, which is important in limb morphogenesis, and may play a role in the development of idiopathic talipes equinovarus. We genotyped SNPs spanning seven apoptotic genes-Casp3, Casp8, Casp9, Casp10, Bid, Bcl-2 and Apaf1-in 210 simplex trios and 139 multiplex families and tested for link-age and association to idiopathic talipes equinovarus. One SNP in each of the genes provided suggestive evidence of association with idiopathic talipes equinovarus. Several haplotypes constructed from these SNPs displayed altered transmission. These data suggest genetic variation in apoptotic genes may play a role in development of idiopathic talipes equinovarus.

OBJECTIVE

This study was conducted to determine the impact of potentially functional polymorphisms in the CASPASE (CASP) genes on the survival of early-stage non-small-cell lung cancer (NSCLC) patients.

METHODS

Four hundred eleven consecutive patients with surgically resected NSCLC were enrolled. Nine potentially functional polymorphisms in the CASP3, CASP7, CASP8, CASP9, and CASP10 genes were investigated. The genotype and haplotype associations with overall survival (OS) and disease-free survival (DFS) were analyzed.

RESULTS

Patients with the rs2227310 GG genotype had a significantly decreased OS and DFS compared with patients with the CC + CG genotype (adjusted hazard ratio [aHR] for OS, 1.67; 95% CI, 1.19 to 2.35; P = .003; aHR for DFS, 1.62; 95% CI, 1.19 to 2.22; P = .002). The rs4645981C>T genotype also had a significant effect on OS and DFS (under a recessive model; aHR for OS, 2.00; 95% CI, 1.04 to 3.85; P = .04; aHR for DFS, 2.76; 95% CI, 1.58 to 4.80; P = .0003). When the rs2227310 and rs4645981 genotypes were combined, patients with one or two bad genotypes had worse OS and DFS compared with those who had zero bad genotypes (aHR for OS, 1.75; 95% CI, 1.25 to 2.45; P = .001; aHR for DFS, 1.66; 95% CI, 1.23 to 2.26; P = .001).

CONCLUSIONS

The CASP7 rs2227310 and CASP9 rs4645981 polymorphisms may affect survival in early-stage NSCLC. The analysis of these polymorphisms can help identify patients at high risk for a poor disease outcome.

Methylation of promoter regions of CpG-rich sites is an important mechanism for silencing of tumor suppressor genes (TSG). To evaluate the role of tumor suppressor genes caspase-8 (CASP8), TIMP-3, E-cadherin (CDH1), p16INK4A, and MGMT in medulloblastoma tumorigenesis, 51 medulloblastomas (46 primary tumor specimens, 5 cell lines) were screened for methylation of promoter linked CpG-islands. For CASP8, we examined the 5' UTR region that has been shown to be associated with expression of CASP8. As detected by methylation specific PCR, methylation rate was low for TIMP-3 (3% of tumor samples; 1/5 cell lines), for MGMT (0% of tumor samples; 1/5 cell lines), for p16INK4A (2% of tumor samples; 2/5 cell lines) and for CDH1 (8% of tumor samples; 1/4 cell lines). CASP8, however, was methylated in 90% of tumor samples and 4/5 cell lines examined. Screening other tumor entities for CASP8 methylation, we found a similarly high level in 6 neuroblastoma cell lines in contrast to 5 osteosarcoma-, 4 Ewing's sarcoma- and 6 non-embryonic tumor cell lines without any increased promoter methylation. From our results we conclude that methylation of the CASP8 5' UTR region may play a role in inactivation of CASP8 in neural crest tumors.

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.

Intrinsically disordered regions serve as molecular recognition elements, which play an important role in the control of many cellular processes and signaling pathways. It is useful to be able to predict positions of disordered residues and disordered regions in protein chains using protein sequence alone. A new method (IsUnstruct) based on the Ising model for prediction of disordered residues from protein sequence alone has been developed. According to this model, each residue can be in one of two states: ordered or disordered. The model is an approximation of the Ising model in which the interaction term between neighbors has been replaced by a penalty for changing between states (the energy of border). The IsUnstruct has been compared with other available methods and found to perform well. The method correctly finds 77% of disordered residues as well as 87% of ordered residues in the CASP8 database, and 72% of disordered residues as well as 85% of ordered residues in the DisProt database.

We have previously reported that the key apoptosis related gene caspase 8 (CASP8) is frequently silenced in small cell lung cancer (SCLC) tumors and cell lines usually, but not always, by aberrant promoter methylation. Because CASP8 is a key component of the death-inducing signaling complex (DISC) when specific death receptors (including DR4, DR5, FAS) are activated by their specific ligands (TRAIL/FASL), we examined expression of the components of the DISC complex in lung cancer cell lines. MYC family members are frequently amplified (MYC+ve) in SCLC, and MYC is a potent inducer of apoptosis. We examined 34 SCLC lines (12 of which were MYC+ve) and 22 NSCLC lines. CASP8 gene expression was frequently lost (79%) at message and protein levels in SCLC but not in non-SCLC (NSCLC). MYC amplification was present in 45% of SCLC cell lines, which had lost CASP8 expression, but not in any of the CASP8 positive lines. The frequency of CASP8 loss was significantly higher in MYC+ve SCLC compared to MYC-ve SCLC or in NSCLC. Analyses of other DISC components showed significantly higher rates of loss of expression of CASP10, DR5, FAS and FASL in SCLC compared to NSCLC. The loss of expression of proapoptotic DISC components was significantly higher in MYC+ve SCLC cell lines and these lines were completely resistant to TRAIL. Expression of CASP10 (a caspase closely related to CASP8) was frequently absent at the protein level in both SCLC and NSCLC lines. Expression of c-FLIP (proteolytically inactive homolog of CASP8) was inversely related to expression of CASP8. Our major conclusions are: (a) The death receptor pathway is differently inactivated at multiple levels in lung cancer cell lines; and (b) MYC amplification in SCLC is associated with inactivation of most components of the DISC complex, with resistance to TRAIL and with expression of c-FLIP. These findings may have considerable clinical and therapeutic implications.

BACKGROUND

Endothelial cells (EC) cultured under altered gravity conditions show a cytoskeletal disorganization and differential gene expression (short-term effects), as well as apoptosis in adherently growing EC or formation of tubular 3D structures (long-term effects).

METHODS

Investigating short-term effects of real microgravity, we exposed EC to parabolic flight maneuvers and analysed them on both protein and transcriptional level. The effects of hypergravity and vibration were studied separately.

RESULTS

Pan-actin and tubulin proteins were elevated by vibration and down-regulated by hypergravity. β-Actin was reduced by vibration. Moesin protein was reduced by both vibration and hypergravity, ezrin potein was strongly elevated under vibration. Gene expression of ACTB, CCND1, CDC6, CDKN1A, VEGFA, FLK-1, EZR, ITBG1, OPN, CASP3, CASP8, ANXA2, and BIRC5 was reduced under vibration. With the exception of CCNA2, CCND1, MSN, RDX, OPN, BIRC5, and ACTB all investigated genes were downregulated by hypergravity. After one parabola (P) CCNA2, CCND1, CDC6, CDKN1A, EZR, MSN, OPN, VEGFA, CASP3, CASP8, ANXA1, ANXA2, and BIRC5 were up-, while FLK1 was downregulated. EZR, MSN, OPN, ANXA2, and BIRC5 were upregulated after 31P.

CONCLUSIONS

Genes of the cytoskeleton, angiogenesis, extracellular matrix, apoptosis, and cell cycle regulation were affected by parabolic flight maneuvers. We show that the microgravity stimulus is stronger than hypergravity/vibration.

The BRAFV600E mutation occurs in approximately 8% of human colorectal cancers and is associated with therapeutic resistance that is due, in part, to reactivation of MEK/ERK signaling cascade. Recently, pathway analysis identified cyclin-dependent kinase 1 (CDK1) upregulation in a subset of human BRAFV600E colorectal cancers. Therefore, it was determined whether CDK1 antagonism enhances the efficacy of MEK inhibition in BRAFV600E colorectal cancer cells. BRAFV600E colorectal cancer cell lines expressing CDK1 were sensitized to apoptosis upon siRNA knockdown or small-molecule inhibition with RO-3306 (CDK1 inhibitor) or dinaciclib (CDK1, 2, 5, 9 inhibitors). Combination of RO-3306 or dinaciclib with cobimetinib (MEK inhibitor) cooperatively enhanced apoptosis and reduced clonogenic survival versus monotherapy. Cells isogenic or ectopic for BRAFV600E displayed resistance to CDK1 inhibitors, as did cells with ectopic expression of constitutively active MEK CDK1 inhibitors induced a CASP8-dependent apoptosis shown by caspase-8 restoration in deficient NB7 cells that enhanced dinaciclib-induced CASP3 cleavage. CDK inhibitors suppressed pro-CASP8 phosphorylation at S387, as shown by drug withdrawal, which restored p-S387 and increased mitosis. In a colorectal cancer xenograft model, dinaciclib plus cobimetinib produced significantly greater tumor growth inhibition in association with a caspase-dependent apoptosis versus either drug alone. The Cancer Genome Atlas (TCGA) transcriptomic dataset revealed overexpression of CDK1 in human colorectal cancers versus normal colon. Together, these data establish CDK1 as a novel mediator of apoptosis resistance in BRAFV600E colorectal cancers whose combined targeting with a MEK/ERK inhibitor represents an effective therapeutic strategy.Implications: CDK1 is a novel mediator of apoptosis resistance in BRAFV600E colorectal cancers whose dual targeting with a MEK inhibitor may be therapeutically effective. Mol Cancer Res; 16(3); 378-89. ©2017 AACR.

The critical assessment of protein structure prediction experiment is a blind assessment of the prediction of protein structure and related topics including function prediction. We present our results in the function/binding site prediction category. Our approach to identify binding sites combined the use of the predicted structure of the targets with both residue conservation and the location of ligands bound to homologous structures. We obtained an average coverage of 83% and 56% accuracy. Analysis of our predictions suggests that over-prediction reduces the accuracy obtained due to large areas of conservation around the binding site that do not bind the ligand. In some proteins such conserved residues may have a functional role. A server version of our method will soon be available.

Oral squamous cell carcinomas (OSCC) are a heterogeneous group of cancers arising from the mucosal lining of the oral cavity. A majority of these cancers are associated with lifestyle risk habits including smoking, excessive alcohol consumption and betel quid chewing. Cetuximab, targeting the epidermal growth factor receptor was approved for the treatment of OSCC in 2006, and remains the only molecular targeted therapy available for OSCC. Here, we reviewed the current findings from genomic analyses of OSCC and discuss how these studies inform on the biological mechanisms underlying OSCC. Exome sequencing revealed that the significantly mutated genes are mainly tumour suppressors. Mutations in FAT1, CASP8, CDKN2A, and NOTCH1 are more frequently found in OSCC when compared to non-OSCC head and neck cancers and other squamous cell carcinomas, and HRAS and PIK3CA are the only significantly mutated oncogenes. The distribution of these mutations also differs in populations with distinct risk habits. Gene expression-based molecular classification showed that OSCC can be divided into distinct subtypes and these have a preferential response to different types of therapies, suggesting that these classifications could have clinical implications. More recently, with the approval of checkpoint inhibitors for the treatment of cancers including OSCC, genomics studies also dissected the genetic signatures of the immune compartment to delineate immune-active and -exhausted subtypes that could inform on the immune status of OSCC patients and guide the development of novel therapies to improve response to immunotherapy. Taken together, genomics studies are informing on the biology of both the epithelial and stromal compartments underlying OSCC development, and we discuss the opportunities and challenges in using these to derive clinical benefit for OSCC patients.

Apoptosis plays a role in the elimination of DNA-damaged cells thus protecting the host from cancer development. Some data indicate that normal variations within the sequence of apoptotic genes may lead to suboptimal apoptotic capacity and therefore increased cancer risk. We tested 19 coding apoptotic gene SNPs in 2-stage molecular epidemiological study. For the preliminary sorting of SNP candidates, we employed a "comparison of extremes" approach, where 111 patients with highly pronounced LC susceptibility (non-smokers or young-onset light smokers) were analyzed against 110 subjects with the evidence for LC tolerance (elderly tumor-free heavy smokers). Three genotypes demonstrated possible association with LC risk (Leu/Leu-homozygotes for Casp5 Val318Leu versus other genotypes: OR=2.47 (95% CI: 1.07-5.69), p=0.03; His-carriers for Casp8 His302Asp: OR=2.26 (95% CI: 1.18-4.31), p=0.02; Arg-carriers for DR4 Lys441Arg: OR=1.89 (95% CI: 1.05-3.40), p=0.03), and therefore were selected for the validation. The extended study included 2 case-control series, namely subjects from Russia (351 LC cases and 538 controls) and Moldova (296 LC cases and 295 controls). Interestingly, all three candidate genotypes consistently demonstrated OR above 1 both in Russian and in Moldovian groups. Although the combined Mantel-Haenszel analysis yet failed to reach statistical significance (OR=1.22 (95% CI: 0.90-1.65), p=0.21; OR=1.17 (95% CI: 0.92-1.50), p=0.21; OR=1.19 (95% CI: 0.95-1.51), p=0.14, respectively), the obtained data indicate that Casp5, Casp8 and DR4 gene polymorphisms may deserve consideration in large-scale case-control studies of LC risk modifiers.

We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24-hour time period relative to the uninfected controls. Of approximately 47,000 genes analyzed, 3,435 were significantly up- or downregulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes asso-ciated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2 and CASP8) and genes that act via the NFĸB pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2 and RELA, IL1B, CAST, CDK2,GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that the X-linked inhibitor of apoptosis protein remained abundant in polymorphonuclear leukocytes over 48 h of LVS infection, whereas BAX mRNA and protein were progressively downregulated. These data strongly suggest that antiapoptotic and prosurvival mechanisms collaborate to sustain the viability of F. tularensis--infected neutrophils.

We present a method to predict the solvent accessibility of proteins which is based on a nearest neighbor method applied to the sequence profiles. Using the method, continuous real-value prediction as well as two-state and three-state discrete predictions can be obtained. The method utilizes the z-score value of the distance measure in the feature vector space to estimate the relative contribution among the k-nearest neighbors for prediction of the discrete and continuous solvent accessibility. The Solvent accessibility database is constructed from 5717 proteins extracted from PISCES culling server with the cutoff of 25% sequence identities. Using optimal parameters, the prediction accuracies (for discrete predictions) of 78.38% (two-state prediction with the threshold of 25%), 65.1% (three-state prediction with the thresholds of 9 and 36%), and the Pearson correlation coefficient (between the predicted and true RSA's for continuous prediction) of 0.676 are achieved An independent benchmark test was performed with the CASP8 targets where we find that the proposed method outperforms existing methods. The prediction accuracies are 80.89% (for two state prediction with the threshold of 25%), 67.58% (three-state prediction), and the Pearson correlation coefficient of 0.727 (for continuous prediction) with mean absolute error of 0.148. We have also investigated the effect of increasing database sizes on the prediction accuracy, where additional improvement in the accuracy is observed as the database size increases. The SANN web server is available at http://lee.kias.re.kr/~newton/sann/.

During an immune response, specific recognition of microbial and tumor antigens leads to the rapid proliferation of lymphocytes. Once the immunological challenge is eliminated, the vast majority of these lymphocytes must be removed via apoptosis. Cell death is also vital for the deletion of autoreactive or chronically activated lymphocytes to prevent the development of autoimmunity in the host. Such processes are highly dependent on death receptors (DRs), molecules of the TNF receptor family. While these DRs promote apoptosis, interference with DR signaling paradoxically interferes with rapid lymphocyte proliferation. Recently, we discovered that T cells lacking Fas-Associated protein with Death Domain (FADD) or caspase-8 (casp8) function, both essential for DR-induced apoptosis, succumb to hyperactivation of autophagy and die through a nonapoptotic form of cell death rather than proliferating after mitogen stimulation. We observed recruitment of FADD, casp8 and serine/threonine kinase RIPK1 to complexes containing Atg5, Atg12 and Atg16L, suggesting that the generation of early autophagosomes leads to the assembly of complexes that activate casp8. Because blockade of RIPK1 or interference with autophagic signaling inhibited this alternative death process, we propose that hyperactive autophagy induced in the absence of caspase activity leads to a necrosis-like form of death that depends on RIPK1 enzymatic function. Herein, we summarize these findings and speculate on the significance and means by which autophagy is normally activated in proliferating lymphocytes.

Neuroblastoma (NB) is a solid tumor of infancy that presents a high rate of spontaneous regression, a phenomenon that likely reflects the activation of an apoptotic/differentiation program. Indeed, the level of expression of molecules involved in the regulation of apoptosis, such as p73 or survivin, is a prognostic factor in NB patients. The caspase-8 gene (CASP8) encodes a key enzyme at the top of the apoptotic cascade. Although methylation of a putative regulatory region of the CASP8 gene reportedly inhibits its transcription in some MYCN-amplified NB, our results indicate that the transcriptional inactivation of caspase-8 occurs in a subset of primary NB independently of MYCN amplification or CpG methylation. In addition, the apoptotic agent fenretinide (4HPR) and interferon-gamma (IFN-gamma) induce caspase-8 expression without modifying the methylation status of this gene. Nevertheless, the methylation level of CASP8 intragenic and promoter regions is higher in MYCN-amplified tumors as compared to nonamplified samples. This phenomenon might reflect the existence of distinct DNA methylation errors in MYCN-amplified and MYCN-single copy tumors. To gain information on the mechanisms that regulate the expression of this crucial apoptotic gene, we searched for potential CASP8 regulatory regions and cloned a DNA element at the 5' terminus of this gene that functionally acts as a promoter only in NB cell lines that express caspase-8. The retinoic acid analogue 4HPR, IFN-gamma, and the demethylating agent 5-aza-cytidine activate this promoter in NB cells that lack endogenous caspase-8, indicating that this element may regulate both constitutive and inducible CASP8 expression. These results indicate also that demethylation of the cellular genome may upregulate CASP8 through the action of trans-acting factors. Our results provide new insights to the regulation of CASP8, a gene with an essential role in a variety of physiologic and pathologic conditions.

Caspase 8 (CASP8) is a key regulator of apoptosis or programmed cell death, and, hence, a defense against cancer. We tested the hypothesis that the CASP8 polymorphism D302H influences risk of glioma through analysis of five series of glioma case patients and controls (n = 1,005 and 1,011, respectively). Carrier status for the rare allele of D302H was associated with a 1.37-fold increased risk (95% confidence interval, 1.10-1.70; P = 0.004). The association of CASP8 D302H with glioma risk indicates the importance of inherited variation in the apoptosis pathway in susceptibility to this form of primary brain tumor.

In an ongoing screen for DNA sequence variants that confer risk of cutaneous basal cell carcinoma (BCC), we conduct a genome-wide association study (GWAS) of 24,988,228 SNPs and small indels detected through whole-genome sequencing of 2,636 Icelanders and imputed into 4,572 BCC patients and 266,358 controls. Here we show the discovery of four new BCC susceptibility loci: 2p24 MYCN (rs57244888[C], OR=0.76, P=4.7 × 10(-12)), 2q33 CASP8-ALS2CR12 (rs13014235[C], OR=1.15, P=1.5 × 10(-9)), 8q21 ZFHX4 (rs28727938[G], OR=0.70, P=3.5 × 10(-12)) and 10p14 GATA3 (rs73635312[A], OR=0.74, P=2.4 × 10(-16)). Fine mapping reveals that two variants correlated with rs73635312[A] occur in conserved binding sites for the GATA3 transcription factor. In addition, expression microarrays and RNA-seq show that rs13014235[C] and a related SNP rs700635[C] are associated with expression of CASP8 splice variants in which sequences from intron 8 are retained.

BACKGROUND

The causes, modes, biological role and prospective significance of cell death in preimplantation development in humans and other mammals are still poorly understood. Early bovine embryos represent a very attractive experimental model for the investigation of this fundamental and important issue.

RESULTS

To obtain reference data on the temporal and spatial occurrence of cell death in early bovine embryogenesis, three-dimensionally preserved embryos of different ages and stages of development up to hatched blastocysts were examined in toto by confocal laser scanning microscopy. In parallel, transcript abundance profiles for selected apoptosis-related genes were analyzed by real-time reverse transcriptase-polymerase chain reaction. Our study documents that in vitro as well as in vivo, the first four cleavage cycles are prone to a high failure rate including different types of permanent cell cycle arrest and subsequent non-apoptotic blastomere death. In vitro produced and in vivo derived blastocysts showed a significant incidence of cell death in the inner cell mass (ICM), but only in part with morphological features of apoptosis. Importantly, transcripts for CASP3, CASP9, CASP8 and FAS/FASLG were not detectable or found at very low abundances.

CONCLUSIONS

In vitro and in vivo, errors and failures of the first and the next three cleavage divisions frequently cause immediate embryo death or lead to aberrant subsequent development, and are the main source of developmental heterogeneity. A substantial occurrence of cell death in the ICM even in fast developing blastocysts strongly suggests a regular developmentally controlled elimination of cells, while the nature and mechanisms of ICM cell death are unclear. Morphological findings as well as transcript levels measured for important apoptosis-related genes are in conflict with the view that classical caspase-mediated apoptosis is the major cause of cell death in early bovine development.

BACKGROUND

Currently, the best characterized genetic aberration in neuroblastoma (NB) is MYCN amplification, which has been clearly related to prognosis. In the present study, we investigated whether specific epigenetic alterations are associated with stage of disease.

METHODS

Sixty-two NBs (45 primary tumors and 17 NBs at relapse) were studied in terms of the methylation status of 19 genes (p15INK4a, p16INK4a, p14ARF, APC, RB1, RASSF1A, BLU, FHIT, RARbeta, INI1, TIMP3, NF2, MGMT, DAPK, FLIP, ECAD, CASP8, and the receptors DcR1 and DcR2).

RESULTS

At diagnosis, we found hypermethylation of RASSF1A in 93% of these tumors, hypermethylation of TIMP3 in 51%, of CASP8 in 38%, of BLU in 34%, of DcR2 in 25%, and of DcR1 in 11%. All 17 tumors tested at relapse showed hypermethylation of RASSF1A (100%), while 10 showed hypermethylation of TIMP3 (59%), six of CASP8 (35%), five of DcR2 (29%), four of BLU (24%), and three of DcR1 (18%). Hypermethylation was related to clinical stage; NBs at stages 1, 2, and 4s were less frequently methylated than stages 3 and 4 disease (P = 0.002).

CONCLUSIONS

These results from our series indicate that hypermethylation of tumor-suppressor genes may be important in the development and evolution of NB. These epigenetic alterations could be used as a marker of the disease and genes regulating methylation should be considered as possible therapeutic targets in NB.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease marked by frequent recurrence and metastasis and stagnant survival rates. To enhance molecular knowledge of HNSCC and define a non-coding RNA (ncRNA) landscape of the disease, we profiled the transcriptome-wide dysregulation of long non-coding RNA (lncRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA) using RNA-sequencing data from 422 HNSCC patients in The Cancer Genome Atlas (TCGA). 307 non-coding transcripts differentially expressed in HNSCC were significantly correlated with patient survival, and associated with mutations in TP53, CDKN2A, CASP8, PRDM9, and FBXW7 and copy number variations in chromosomes 3, 5, 7, and 18. We also observed widespread ncRNA correlation to concurrent TP53 and chromosome 3p loss, a compelling predictor of poor prognosis in HNSCCs. Three selected ncRNAs were additionally associated with tumor stage, HPV status, and other clinical characteristics, and modulation of their expression in vitro reveals differential regulation of genes involved in epithelial-mesenchymal transition and apoptotic response. This comprehensive characterization of the HNSCC non-coding transcriptome introduces new layers of understanding for the disease, and nominates a novel panel of transcripts with potential utility as prognostic markers or therapeutic targets.

Epigenetic aberrations and a CpG island methylator phenotype are associated with poor outcome in children with neuroblastoma (NB). Previously, we have shown that valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, exerts antitumor effects in an NB xenograft model. However, the underlying antitumor molecular mechanisms are largely unknown. In this study, we examined the role of HDAC in cell proliferation, cell cycle progression, gene expression patterns, and epigenome in NB. Cell proliferation, cell cycle progression, caspase activity, RNA and protein expression, quantitative methylation, and global DNA methylation were examined in NBL-W-N and LA1-55n NB cell lines. Our studies showed that inhibition of HDAC decreased NB proliferation, and induced caspase activity and G1 growth arrest. Expression patterns of cancer-related genes were modulated by VPA. The expression of THBS1, CASP8, SPARC, CDKN1A, HIC1, CDKN1B, and HIN1 was upregulated, and that of MYCN and TIG1 was downregulated. HDAC inhibition decreased methylation levels of THBS1 and RASSF1A promoters. Inhibition of HDAC increased acetylation of histone 4 and overall DNA methylation levels. Our studies showed that inhibition of HDAC blocked cell proliferation and cell cycle progression in relation to alteration in cancer-related genes, increased overall DNA methylation, and decreased methylation of tumor suppressor genes. Further studies examining the antitumor effects of VPA in NB are warranted.

Caspase-8 (CASP8) is a protease that initiates apoptosis and regulates inflammation and immune responses. We identified germline mutations in CASP8 in 3 unrelated patients with infant-onset inflammatory bowel disease: 2 patients were homozygous for the mutation 710A>G, p.Q237R, which resulted in reduced protein expression, and 1 patient carried the mutation 793C>T, p.R265W. We isolated peripheral blood mononuclear cells from our index patient and observed defects in T- and B-cell maturation, proliferation, and/or activation. Macrophages from 1 patient with CASP8 deficiency and monocytic BLaER1 cells with knockout of CASP8 or overexpression of CASP8 with the 710A>G mutation had altered inflammasome activity on stimulation with lipopolysaccharide. Patient-derived intestinal organoids and colon carcinoma cells with knockout of CASP8 had defects in cell death processes that involved loss of TRAIL signaling and increased necroptosis. These findings indicate that CASP8 controls inflammation, innate and adaptive immunity, and intestinal barrier integrity in humans.

Thyroid cancer is the most common endocrine cancer with 1,690 deaths each year. There are four main types of which the papillary and follicular types together account for >90% followed by medullary cancers with 3% to 5% and anaplastic carcinomas making up <3%. Epigenetic events of DNA hypermethylation are emerging as promising molecular targets for cancer detection. Our immediate and long term goal is to identify DNA methylation markers for early detection of thyroid cancer. This pilot study comprised of 21 patients to include 11 papillary thyroid cancers (PTC), 2 follicular thyroid cancers (FTC), 5 normal thyroid cases, and 3 hyperthyroid cases. Aberrant promoter methylation was examined in 24 tumor suppressor genes using the methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) assay and in the NIS gene using methylation-specific PCR (MSP). The frequently methylated genes were CASP8 (17/21), RASSF1 (16/21) and NIS (9/21). In the normal samples, CASP8, RASSF1 and NIS were methylated in 5/5, 4/5 and 1/5 respectively. In the hyperthyroid samples, CASP8, RASSF1 and NIS were methylated in 3/3, 2/3 and 1/3 respectively. In the thyroid cancers, CASP8, RASSF1, and NIS were methylated in 9/13, 10/13, and 7/13 respectively. CASP8, RASSF1 and NIS were also methylated in concurrently present normal thyroid tissue in 3/11, 4/11 and 3/11 matched thyroid cancer cases (matched for presence of both normal thyroid tissue and thyroid cancer), respectively. Our data suggests that aberrant methylation of CASP8, RASSF1, and NIS maybe an early change in thyroid tumorigenesis regardless of cell type.