BACKGROUND

Few high penetrance genes are known in Malignant Melanoma (MM), however, the involvement of low-penetrance genes such as MC1R, OCA2, ASIP, SLC45A2 and TYR has been observed. Lately, genome-wide association studies (GWAS) have been the ideal strategy to identify new common, low-penetrance susceptibility loci. In this case-control study, we try to validate in our population nine melanoma associated markers selected from published GWAS in melanoma predisposition.

METHODS

We genotyped the 9 markers corresponding to 8 genes (PARP1, MX2, ATM, CCND1, NADSYN1, CASP8, IRF4 and CYP2R1) in 566 cases and 347 controls from a Spanish population using KASPar probes. Genotypes were analyzed by logistic regression and adjusted by phenotypic characteristics.

RESULTS

We confirm the protective role in MM of the rs3219090 located on the PARP1 gene (p-value 0.027). Additionally, this SNP was also associated with eye color (p-value 0.002). A second polymorphism, rs12203592, located on the IRF4 gene was associated with protection to develop MM for the dominant model (p-value 0.037). We have also observed an association of this SNP with both lentigines (p-value 0.014) and light eye color (p-value 3.76 × 10(-4)). Furthermore, we detected a novel association with rs1485993, located on the CCND1 gene, and dark eye color (p-value 4.96 × 10(-4)). Finally, rs1801516, located on the ATM gene, showed a trend towards a protective role in MM similar to the one firstly described in a GWAS study.

CONCLUSIONS

To our knowledge, this is the first time that these SNPs have been associated with MM in a Spanish population. We confirmed the proposed role of rs3219090, located on the PARP1 gene, and rs12203592, located on the IRF4 gene, as protective to MM along the same lines as have previous genome-wide associated works. Finally, we have seen associations between IRF4, PARP1, and CCND1 and phenotypic characteristics, confirming previous results for the IRF4 gene and presenting novel data for the last two, suggesting that pigmentation characteristics correlated with eye color are potential mediators between PARP1 and MM protection.

Amyloid-beta (Aβ) peptides, Aβ 1-42 (Aβ42) and Aβ43 in particular, cause neurotoxicity and cell death in the brain of Alzheimer's disease (AD) at higher concentrations. Carnosic acid (CA), a phenolic diterpene compound in the labiate herbs rosemary and sage, serves as an activator for neuroprotective and neurotrophic functions in brain cells. We investigated the effect of CA on apoptosis induced by Aβ42 or Aβ43 in cultured SH-SY5Y human neuroblastoma cells. Treatment of the cells with Aβ42 or Aβ43 (monomer, 10 μM each) induced apoptosis, which was confirmed by the cleavage of poly-(ADP-ribose) polymerase (PARP) and apoptosis-inducing factor (AIF). Concurrently, the Aβ treatment induced the activation of caspase (Casp) cascades including an effector Casp (Casp3) and initiator Casps (Casp4, Casp8 and Casp9). Pretreatment of the cells with CA (10 μM) partially attenuated the apoptosis induced by Aβ42 or Aβ43. CA pretreatment also reduced the cellular oligomers of Aβ42 and Aβ43. These results suggest that CA suppressed the activation of Casp cascades by reducing the intracellular oligomerization of exogenous Aβ42/43 monomer. The ingestion of an adequate amount of CA may have a potential in the prevention of Aβ-mediated diseases, particularly AD.

Caspase-8 (CASP8) plays a critical role in regulating apoptosis, and its functional polymorphisms may modify cancer risk. We investigated the possible association between CASP8 -652 6N ins/del (rs3834129) and the risk of breast cancer in a sample of Iranian population. This case-control study was done on 236 breast cancer patients and 203 cancer free healthy female. We designed a rapid and simple bi-directional PCR allele-specific amplification (bi-PASA) for detection of CASP8 -652 6N ins/del polymorphism. The results showed that the CASP8 -652 6N del/dl genotype was inversely associated with breast cancer risk (OR=0.33, 95% CI=0.17-0.65, p=0.001). The frequencies of the del allele in cases and controls were 29.1% and 38.6%, respectively. An inverse association between CASP8 6N del variant and the risk of breast cancer (OR=0.66, 95% CI=0.66-0.87, p=0.002) was found. In conclusion, the result suggests that the CASP8 -652 6N del polymorphism plays a protective role in susceptibility to breast cancer in our population. Further studies in other populations with larger samples are needed to confirm these findings.

BACKGROUND

Although non muscle invasive bladder cancer (NMIBC) generally has a good long-term prognosis, up to 80% of patients will nevertheless experience local recurrence after the primary tumor resection. The search for markers capable of accurately identifying patients at high risk of recurrence is ongoing. We retrospectively evaluated the methylation status of a panel of 24 tumor suppressor genes (TIMP3, APC, CDKN2A, MLH1, ATM, RARB, CDKN2B, HIC1, CHFR, BRCA1, CASP8, CDKN1B, PTEN, BRCA2, CD44, RASSF1, DAPK1, FHIT, VHL, ESR1, TP73, IGSF4, GSTP1 and CDH13) in primary lesions to obtain information about their role in predicting local recurrence in NMIBC.

METHODS

Formaldehyde-fixed paraffin-embedded (FFPE) samples from 74 patients operated on for bladder cancer were analyzed by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA): 36 patients had relapsed and 38 were disease-free at the 5-year follow up. Methylation status was considered as a dichotomous variable and genes showing methylation ≥20% were defined as "positive".

RESULTS

Methylation frequencies were higher in non recurring than recurring tumors. A statistically significant difference was observed for HIC1 (P = 0.03), GSTP1 (P = 0.02) and RASSF1 (P = 0.03). The combination of the three genes showed 78% sensitivity and 66% specificity in identifying recurrent patients, with an overall accuracy of 72%.

CONCLUSIONS

Our preliminary data suggest a potential role of HIC1, GSTP1 and RASSF1 in predicting local recurrence in NMIBC. Such information could help clinicians to identify patients at high risk of recurrence who require close monitoring during follow up.

BACKGROUND

Recent candidate and genome-wide association studies have identified variants altering susceptibility to breast cancer.

OBJECTIVE

To establish the relevance of these variants to breast cancer risk in familial breast cancer cases both with and without BRCA1 or BRCA2 (BRCA1/2) mutations.

METHODS

A cohort of unrelated individuals with breast cancer due to the presence of either BRCA1 (121) or BRCA2 mutations (109) and individuals with familial breast cancer not due to BRCA1/2 mutations (722) were genotyped using Taqman SNP Genotyping Assays. Allele frequencies were compared with an ethnically and gender-matched group (436).

RESULTS

A synonymous variant (Ser51) in TOX3 (previously TNRC9) was associated with an increased risk of breast cancer (OR=1.82, p<0.001) in BRCA2 mutation carriers. The associations for FGFR2 (OR=1.20, p=0.046), TOX3 (OR=1.5, p<0.001), MAP3K1 (OR=1.26 p=0.03), CASP8 (OR=0.73 p=0.02) and the chromosome 8-associated SNP (OR=1.31, p=0.004) were replicated in individuals without BRCA1/2 mutations. In addition, homozygote carriers of MAP3K1 variants were shown to have a significantly lower Manchester Score (mean 13.8-17.6, p=0.003), whereas individuals carrying one or two copies of the FGFR2 variant had a higher Manchester Score (mean 17.5-17.9, p=0.01).

CONCLUSIONS

This study confirms that susceptibility variants in FGFR2, TOX3 and MAP3K1 and on chromosome 8q are all associated with increased risk of cancer in individuals with a family history of breast cancer, whereas CASP8 is protective in this context. The level of risk is dependent on the strength of the family history and the presence of a BRCA1/2 mutation and contributes to the understanding of the use of these variants in clinical risk prediction.

Protein structure refinement is an important but unsolved problem; it must be solved if we are to predict biological function that is very sensitive to structural details. Specifically, critical assessment of techniques for protein structure prediction (CASP) shows that the accuracy of predictions in the comparative modeling category is often worse than that of the template on which the homology model is based. Here we describe a refinement protocol that is able to consistently refine submitted predictions for all categories at CASP7. The protocol uses direct energy minimization of the knowledge-based potential of mean force that is based on the interaction statistics of 167 atom types (Summa and Levitt, Proc Natl Acad Sci USA 2007; 104:3177-3182). Our protocol is thus computationally very efficient; it only takes a few minutes of CPU time to run typical protein models (300 residues). We observe an average structural improvement of 1% in GDT_TS, for predictions that have low and medium homology to known PDB structures (Global Distance Test score or GDT_TS between 50 and 80%). We also observe a marked improvement in the stereochemistry of the models. The level of improvement varies amongst the various participants at CASP, but we see large improvements (>10% increase in GDT_TS) even for models predicted by the best performing groups at CASP7. In addition, our protocol consistently improved the best predicted models in the refinement category at CASP7 and CASP8. These improvements in structure and stereochemistry prove the usefulness of our computationally inexpensive, powerful and automatic refinement protocol.

Recent large-scale studies have been successful in identifying common, low-penetrance variants associated with common cancers. One such variant in the caspase-8 (CASP8) gene, D302H (rs1045485), has been confirmed to be associated with breast cancer risk, although the functional effect of this polymorphism (if any) is not yet clear. In order to further map the CASP8 gene with respect to breast cancer susceptibility, we performed extensive haplotype analyses using single nucleotide polymorphisms (SNP) chosen to tag all common variations in the gene (tSNP). We used a staged study design based on 3,200 breast cancer and 3,324 control subjects from the United Kingdom, Utah, and Germany. Using a haplotype-mining algorithm in the UK cohort, we identified a four-SNP haplotype that was significantly associated with breast cancer and that was superior to any other single or multi-locus combination (P=8.0 x 10(-5)), with a per allele odds ratio and 95% confidence interval of 1.30 (1.12-1.49). The result remained significant after adjustment for the multiple testing inherent in mining techniques (false discovery rate, q=0.044). As expected, this haplotype includes the D302H locus. Multicenter analyses on a subset of the tSNPs yielded consistent results. This risk haplotype is likely to carry one or more underlying breast cancer susceptibility alleles, making it an excellent candidate for resequencing in homozygous individuals. An understanding of the mode of action of these alleles will aid risk assessment and may lead to the identification of novel treatment targets in breast cancer.

Apoptosis is known to be involved in tumorigenesis and a defective ratio between cell proliferation and apoptosis may contribute to the emergence of a malignant phenotype. Transcriptional silencing of apoptosis-related genes associated with aberrant promoter methylation may impair the apoptotic machinery, ultimately leading to cancer development. Aberrant promoter methylation of numerous genes involved in many different pathways is frequent in prostate cancer. Our aim was to quantitatively assess the methylation status of several apoptosis-related genes in prostate adenocarcinoma (PCa) and its precursor lesion, high-grade prostatic intraepithelial neoplasia (HGPIN). First, 120 PCa and 39 HGPIN were screened for altered expression of BCL2, CASP8, CASP3, DAPK DR3, DR4, DR6, FAS, TMS1, TNFR2, using 28 benign prostate hyperplasias and 10 normal prostates as controls. Underexpressed genes were then assessed by quantitative methylation-specific PCR to determine the promoter methylation status. Finally, quantitative mRNA expression of aberrantly methylated genes was performed and methylation data was correlated with standard clinicopathologic parameters. DAPK, DR4 and TNFR2 were significantly overexpressed in HGPIN and PCa, whereas BCL2, TMS1, and FAS were downregulated. Although methylation levels were significantly higher for TMS1 and BCL2 (correlating with advanced stage), an inverse correlation with mRNA expression was found only for BCL2. We concluded that the apoptotic pathways are largely preserved in prostate carcinogenesis, in particular the extrinsic pathway, with the exception of FAS and TMS1, which are epigenetically downregulated. In addition, BCL2 was also found to be frequently silenced in PCa due to aberrant promoter methylation, thus supporting a future role for apoptosis-targeted therapy in prostate cancer.

Expression analysis of apoptotic genes was performed for 15 patients with acute myelocytic leukemia (AML) at the time of diagnosis to identify genes and signaling pathways involved in the regulation of cell survival and apoptosis during leukemogenesis. cDNA array analysis revealed 34 genes whose expression was significantly different compared to others. Tumor suppressor genes TP53 and CDKN2A were downregulated and protooncogenes JUN and GRB10 were upregulated. Furthermore, several cellular signaling pathways acting either in cell cycle regulation or in apoptosis were altered. Deregulation was found in pathways that contribute to genomic stability (by downregulation of either TP53 or CSE1L and by upregulation of GADD45A) and regulate cell cycle progression (by downregulation of CDKN2A and upregulation of RBBP4, CDC37, and NEDD5). Alterations at the transcriptional level were identified, namely, upregulation of JUN and E2F5. Abnormalities were observed in the regulation of the caspases through upregulation of CASP8 and by altered expression of BCL2-related pathway. Extrinsic apoptotic signals mediated by IGFs were deregulated and the glutathione detoxification pathway was downregulated. These findings provide insight into the regulation of balance between apoptosis and cell proliferation signals, and suggest that these genes and pathways may have an important role in the pathogenesis of AML.

One approach to predict a protein fold from a sequence (a target) is based on structures of related proteins that are used as templates. We present an algorithm that examines a set of candidates for templates, builds from each of the templates an atomically detailed model, and ranks the models. The algorithm performs a hierarchical selection of the best model using a diverse set of signals. After a quick and suboptimal screening of template candidates from the protein data bank, the current method fine-tunes the selection to a few models. More detailed signals test the compatibility of the sequence and the proposed structures, and are merged to give a global fitness measure using linear programming. This algorithm is a component of the prediction server LOOPP (http://www.loopp.org). Large-scale training and tests sets were designed and are presented. Recent results of the LOOPP server in CASP8 are discussed.

In order to be successful CASP experiments require experimentally determined protein structures. These structures form the basis of the experiment. Structural genomics groups have provided the vast majority of these structures in recent editions of CASP. Before the structure prediction assessment can begin these target structures must be divided into structural domains for assessment purposes and each assessment unit must be assigned to one or more tertiary structure prediction categories. In CASP8 target domain boundaries were based on visual inspection of targets and their experimental data, and on superpositions of the target structures with related template structures. As in CASP7 target domains were broadly classified into two different categories: "template-based modeling" and "free modeling." Assessment categories were determined by structural similarity between the target domain and the nearest structural templates in the PDB and by whether or not related structural templates were used to build the models. The vast majority of the 164 assessment units in CASP8 were classified as template-based modeling. Just 10 target domains were defined as free modeling. In addition three targets were assessed in both the free modeling and template based categories and a subset of 50 template-based models was evaluated as part of the "high accuracy" subset. The targets submitted for CASP8 confirmed a trend that has been apparent since CASP5: targets submitted to the CASP experiments are becoming easier to predict.

During the 7th Critical Assessment of Protein Structure Prediction (CASP7) experiment, it was suggested that the real value of predicted residue-residue contacts might lie in the scoring of 3D model structures. Here, we have carried out a detailed reassessment of the contact predictions made during the recent CASP8 experiment to determine whether predicted contacts might aid in the selection of close-to-native structures or be a useful tool for scoring 3D structural models. We used the contacts predicted by the CASP8 residue-residue contact prediction groups to select models for each target domain submitted to the experiment. We found that the information contained in the predicted residue-residue contacts would probably have helped in the selection of 3D models in the free modeling regime and over the harder comparative modeling targets. Indeed, in many cases, the models selected using just the predicted contacts had better GDT-TS scores than all but the best 3D prediction groups. Despite the well-known low accuracy of residue-residue contact predictions, it is clear that the predictive power of contacts can be useful in 3D model prediction strategies.

UNASSIGNED

The accurate ranking of predicted structural models and selecting the best model from a given candidate pool remain as open problems in the field of structural bioinformatics. The quality assessment (QA) methods used to address these problems can be grouped into two categories: consensus methods and single-model methods. Consensus methods in general perform better and attain higher correlation between predicted and true quality measures. However, these methods frequently fail to generate proper quality scores for native-like structures which are distinct from the rest of the pool. Conversely, single-model methods do not suffer from this drawback and are better suited for real-life applications where many models from various sources may not be readily available.

UNASSIGNED

In this study, we developed a support-vector-machine-based single-model global quality assessment (SVMQA) method. For a given protein model, the SVMQA method predicts TM-score and GDT_TS score based on a feature vector containing statistical potential energy terms and consistency-based terms between the actual structural features (extracted from the three-dimensional coordinates) and predicted values (from primary sequence). We trained SVMQA using CASP8, CASP9 and CASP10 targets and determined the machine parameters by 10-fold cross-validation. We evaluated the performance of our SVMQA method on various benchmarking datasets. Results show that SVMQA outperformed the existing best single-model QA methods both in ranking provided protein models and in selecting the best model from the pool. According to the CASP12 assessment, SVMQA was the best method in selecting good-quality models from decoys in terms of GDTloss.

UNASSIGNED

SVMQA method can be freely downloaded from http://lee.kias.re.kr/SVMQA/SVMQA_eval.tar.gz.

UNASSIGNED

jlee@kias.re.kr.

UNASSIGNED

Supplementary data are available at Bioinformatics online.

Caspase 8 (CASP8) is an apoptosis-related cysteine peptidase involved in the death receptor pathway and likely in the mitochondrial pathway. A CASP8 promoter region six-nucleotide deletion/insertion (-652 6N ins/del) variant and a coding region D302H polymorphism are reportedly important in cancer development, but no reported study has assessed the associations of these genetic variations with risk of head and neck cancer. In a hospital-based study of non-Hispanic whites, we genotyped CASP8 -652 6N del and 302H variants in 1,023 patients with squamous cell carcinoma of the head and neck (SCCHN) and 1,052 cancer-free controls. Crude and adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated using unconditional logistic regression models. The CASP8 -652 6N del variant genotypes or haplotypes were inversely associated with SCCHN risk (adjusted OR, 0.70; 95% CI, 0.57-0.85 for the ins/del + del/del genotypes compared with the ins/ins genotype; adjusted OR, 0.73; 95% CI, 0.55-0.97 for the del-D haplotype compared with the ins-D haplotype). Furthermore, the number of the CASP8 -652 6N del (but not 302H) variant allele tended to correlate with increased levels of camptothecin-induced p53-mediated apoptosis in T lymphocytes from 170 cancer-free controls. We concluded that the CASP8 -652 6N del variant allele may contribute to the risk of developing SCCHN in non-Hispanic white populations. Further validation by population-based case-control studies and rigorous mechanistic studies is warranted.

OBJECTIVE

Caspase-8 (CASP8) is a key regulator of apoptosis or programmed cell death, an essential defense mechanism against hyperproliferation and malignancy. We hypothesized that the variants in the CASP8 gene are associated with risk of bladder cancer.

METHODS

In a hospital-based case-control study of 365 case patients with newly diagnosed bladder transitional cell carcinoma and 368 cancer-free controls frequency-matched by age and sex, we genotyped the functional -652 6N ins/del polymorphism (rs3834129) in the promoter of CASP8 and assessed its associations with risk of bladder cancer and interaction with tobacco smoking.

RESULTS

A significant decreased risk of bladder cancer was found for the CASP8 -652 6N ins/del (adjusted odds ratio, 0.72; 95% confidence interval, 0.53-0.99) and del/del (odds ratio, 0.37; 95% confidence interval, 0.18-0.77) genotypes. Furthermore, a significant additive interaction between CASP8 polymorphism and tobacco smoking on bladder cancer risk was observed.

CONCLUSIONS

These results suggested that the CASP8 -652 6N ins/del polymorphism is involved in etiology of bladder cancer and thus may be a marker for genetic susceptibility to bladder cancer in Chinese populations. Larger studies are warranted to validate our findings.

Emerging biological and translational insights from large sequencing efforts underscore the need for genetically-relevant cell lines to study the relationships between genomic alterations of tumors, and therapeutic dependencies. Here, we report a detailed characterization of a novel panel of clinically annotated oral squamous cell carcinoma (OSCC) cell lines, derived from patients with diverse ethnicity and risk habits. Molecular analysis by RNAseq and copy number alterations (CNA) identified that the cell lines harbour CNA that have been previously reported in OSCC, for example focal amplications in 3q, 7p, 8q, 11q, 20q and deletions in 3p, 5q, 8p, 18q. Similarly, our analysis identified the same cohort of frequently mutated genes previously reported in OSCC including TP53, CDKN2A, EPHA2, FAT1, NOTCH1, CASP8 and PIK3CA. Notably, we identified mutations (MLL4, USP9X, ARID2) in cell lines derived from betel quid users that may be associated with this specific risk factor. Gene expression profiles of the ORL lines also aligned with those reported for OSCC. By focusing on those gene expression signatures that are predictive of chemotherapeutic response, we observed that the ORL lines broadly clustered into three groups (cell cycle, xenobiotic metabolism, others). The ORL lines noted to be enriched in cell cycle genes responded preferentially to the CDK1 inhibitor RO3306, by MTT cell viability assay. Overall, our in-depth characterization of clinically annotated ORL lines provides new insight into the molecular alterations synonymous with OSCC, which can facilitate in the identification of biomarkers that can be used to guide diagnosis, prognosis, and treatment of OSCC.

Death receptors of the Tumor Necrosis Factor (TNF) family form membrane-bound self-activating signaling complexes that initiate apoptosis through cleavage of proximal caspases including CASP8 and 10. Here we show that overexpression of the cytoplasmic domain (CD) of the DR4 TRAIL receptor (TNFRSF10A, TRAIL R1) in human breast, lung, and colon cancer cell lines, using an adenovirus vector (Ad-DR4-CD), leads to p53-independent apoptotic cell death involving cleavage of CASP8 and 10 proximally and CASP3, 6, and 7 distally. DR4-CD overexpression also leads to cleavage of poly(ADP-ribose) polymerase (PARP) and the DNA fragmentation factor (DFF45; ICAD). Importantly, normal lung fibroblasts are resistant to DR4-CD overexpression and show no evidence of PARP-, CASP8- or CASP3-cleavage despite similar levels of adenovirus-delivered DR4-CD protein as the cancer cells. These results suggest that DR4 may signal death through known caspases and that further studies are required to evaluate Ad-DR4-CD as a novel anti-cancer agent. Finally, we show that overexpression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (CDKN1A), or its N-terminal 91 amino acids containing cell cycle-inhibitory activity, inhibits DR4-CD-dependent proximal caspase cleavage. The blockage of initiator caspase activation provides a novel insight into how p21 may suppress apoptosis and enhance cell survival.

Multibody potentials have been of much interest recently because they take into account three dimensional interactions related to residue packing and capture the cooperativity of these interactions in protein structures. Our goal was to combine long range multibody potentials and short range potentials to improve recognition of native structure among misfolded decoys. We optimized the weights for four-body nonsequential, four-body sequential, and short range potentials to obtain optimal model ranking results for threading and have compared these data against results obtained with other potentials (26 different coarse-grained potentials from the Potentials 'R'Us web server have been used). Our optimized multibody potentials outperform all other contact potentials in the recognition of the native structure among decoys, both for models from homology template-based modeling and from template-free modeling in CASP8 decoy sets. We have compared the results obtained for this optimized coarse-grained potentials, where each residue is represented by a single point, with results obtained by using the DFIRE potential, which takes into account atomic level information of proteins. We found that for all proteins larger than 80 amino acids our optimized coarse-grained potentials yield results comparable to those obtained with the atomic DFIRE potential.

BACKGROUND

A number of susceptibility genes are common to breast and pancreatic cancer. Recently, several breast cancer susceptibility loci have been identified through genome-wide association studies. Here we evaluated possible associations between these single nucleotide polymorphisms (SNP) and pancreatic cancer risk.

METHODS

Ten SNPs from FGFR2, TOX3, MAP3K1, H19, LSP1, chromosome 8q24, CASP8, and LUM were investigated for associations with pancreatic cancer risk following genotyping in 1,143 Caucasian individuals with pancreatic adenocarcinoma and 1,097 unaffected controls from a clinic-based pancreatic cancer case-control study.

RESULTS

CASP8 rs1045485 [odds ratio (OR), 0.78; 95% confidence interval (95% CI), 0.65-0.9; P = 0.005] and MAP3K1 rs889312 (OR, 0.85; 95% CI, 0.74-0.97; P = 0.017) showed evidence of association with risk of pancreatic cancer. The CASP8 rs1045485 association was evident in ever smokers (P = 0.002), but not in nonsmokers (P = 0.55), and the effect was strongest in heavy smokers (OR, 0.52; 95% CI, 0.29-0.93; P = 0.03). In contrast the MAP3K1 rs889312 association was only evident in nonsmokers (OR, 0.78; 95% CI, 0.64-0.95; P = 0.01). In addition, evaluation of the influence of the 10 SNPs on survival detected significant associations between outcome for locally advanced pancreatic cancer cases and both 8q rs6983561 (P = 0.045) and LUM rs2268578 (P = 0.02).

CONCLUSIONS

Association studies in a large pancreatic case-control study indicate that SNPs associated with breast cancer may also be associated with pancreatic cancer susceptibility and survival.

BACKGROUND

Although caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. As cells of the innate immune system, and in particular macrophages, are now at the forefront of autoimmune disease pathogenesis, we examined the potential involvement of caspase-8 within this population.

METHODS

Cre (LysM) Casp8 (fl/fl) mice were bred via a cross between Casp8 (fl/fl) mice and Cre (LysM) mice, and RIPK3 (-/-) Cre (LysM) Casp8 (fl/fl) mice were generated to assess the contribution of receptor-interacting serine-threonine kinase (RIPK)3. Immunohistochemical and immunofluorescence analyses were used to examine renal damage. Flow cytometric analysis was employed to characterize splenocyte distribution and activation. Cre (LysM) Casp8 (fl/fl) mice were treated with either Toll-like receptor (TLR) agonists or oral antibiotics to assess their response to TLR activation or TLR agonist removal. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure cytokine/chemokine and immunoglobulin levels in serum and cytokine levels in cell culture studies. In vitro cell culture was used to assess macrophage response to cell death stimuli, TLR activation, and M1/M2 polarization. Data were compared using the Mann-Whitney U test.

RESULTS

Loss of caspase-8 expression in macrophages promotes onset of a mild systemic inflammatory disease, which is preventable by the deletion of RIPK3. In vitro cell culture studies reveal that caspase-8-deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8-deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations. Loss of caspase-8 in macrophages dictates the response to TLR activation, as injection of TLR ligands upregulates expression of costimulatory CD86 on the Ly6C(high)CD11b(+)F4/80(+) splenic cells, and oral antibiotic treatment to remove microbiota prevents splenomegaly and lymphadenopathy in Cre (LysM) Casp8 (fl/fl) mice. Further, caspase-8-deficient macrophages are hyperresponsive to TLR activation and exhibit aberrant M1 macrophage polarization due to RIPK activity.

CONCLUSIONS

These data demonstrate that caspase-8 functions uniquely in macrophages by controlling the response to TLR activation and macrophage polarization in an RIPK-dependent manner.

Methyl jasmonate (MJ) is a botanical hormone that serves as a signal transduction intermediate and regulates cell death in stressed plants. MJ induces cell cycle arrest, apoptosis and non-apoptotic cell death selectively in cancer cells. However, the underlying mechanism of MJ-induced apoptosis remains unclear. In this study, we examined the molecular mechanism through which MJ induces apoptosis in human non-small cell lung cancer (NSCLC). We found that MJ triggered apoptosis via the DDIT3-TNFRSF10B-CASP axis. MJ treatment significantly decreased the expression of CFLAR (CASP8 and FADD-like apoptosis regulator, an inhibitor of CASP8) in NSCLC cells, and ectopic expression of CFLAR partly protected cells from MJ-induced apoptosis. MJ also induced pro-apoptotic autophagy in NSCLC cells. Importantly, inhibition of ROS suppressed both MJ-induced apoptosis and autophagy. Taken together, MJ induces apoptosis and pro-apoptotic autophagy in NSCLC cells through the ROS pathway. Thus, MJ and its derivative treatment may serve as a novel chemotherapeutic strategy for cancer therapy.

Studies on genetic changes underlying prostate cancer and the possible signaling pathways are getting increased day by day, and new treatment methods are being searched for. The aim of the present study is to investigate the effects of ferulic acid (FA), a phenolic compound, on cell cycle, apoptosis, invasion, and colony formation in the PC-3 and LNCaP prostate cancer cells. The effect of FA on cell viability was determined via a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) method. Total RNA was isolated with Tri Reagent. Expression of 84 genes for both cell cycle and apoptosis separately was evaluated by reverse transcriptase PCR (RT-PCR). Protein expressions were evaluated by Western blot analysis. Furthermore, apoptotic effects of FA were observed with terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay. Effects of FA on cell invasion and colony formation were determined using Matrigel chamber and colony assay, respectively. The half maximal inhibitory concentration (IC50) dose of FA was found to be 300 μM in PC-3 cells and 500 μM in LNCaP cells. According to RT-PCR results, it was observed that FA inhibited cell proliferation by increasing the gene expressions of ATR, ATM, CDKN1A, CDKN1B, E2F4, RB1, and TP53 and decreasing the gene expressions of CCND1, CCND2, CCND3, CDK2, CDK4, and CDK6 in PC-3 cells. On the other hand, it was seen that FA suppressed cell proliferation by increasing in the gene expressions of CASP1, CASP2, CASP8, CYCS, FAS, FASLG, and TRADD and decreasing in the gene expressions of BCL2 and XIAP in LNCaP cells. In this study, protein expression of CDK4 and BCL2 genes significantly decreased in these cells. It could induce apoptosis in PC-3 and LNCaP cells. Also, it was observed that FA suppressed the invasion in PC-3 and LNCaP cells. Moreover, it suppressed the colony formation. In conclusion, it has been observed that FA may lead to cell cycle arrest in PC-3 cells while it may cause apoptosis in LNCaP cells.

Photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX synthesis as a photosensitizing reagent is an encouraging modality for cancer treatment. Understanding the mechanism of tumor phototoxicity is important to provide a basis for combinatory therapy regimens. A normal cell line (UROtsa, urothelial) and two tumor cell lines (RT4, urothelial; HT29, colonic) were treated with cell line-specific LD50 doses of light after exposure to 5-aminolevulinic acid (100 microg/mL), and harvested for RNA extraction 0, 10, and 30 minutes after irradiation. The RNA was hybridized to the metg001A Affymetrix GeneChip containing 2,800 genes, focusing on cancer-related and growth regulatory targets. Comparing the gene expression profiles between the different samples, 40 genes (e.g., SOD2, LUC7A, CASP8, and DUSP1) were identified as significantly altered in comparison with the control samples, and grouped according to their gene ontology. We selected caspase-8 (CASP8) and dual specificity phosphatase 1 (DUSP1) for further validation of the array findings, and compared their expression with the expression of the immediate early gene FOS by quantitative reverse transcription-PCR. RNA expression of CASP8 stayed unchanged whereas DUSP1 RNA was up-regulated in normal and tumor cells starting 30 minutes after irradiation. In contrast, FOS RNA was found continuously up-regulated over time in all three cell lines. Induction of DUSP1 protein expression was clearly shown after 1 hour using Western blot analysis. Interestingly, no changes of caspase-8 protein expression but activation of catalytic activity was detected only in UROtsa cells starting 1 hour after photodynamic therapy, whereas no changes were seen in both tumor cell lines. According to caspase-8, the active caspase 3 fragment was found only in the normal urothelial cell line (UROtsa) 1 hour after photodynamic therapy. Combined data analysis suggests that photodynamic therapy in vitro (LD50) leads to apoptosis in UROtsa and to necrosis in the tumor cell lines, respectively. RNA expression profiling of normal and tumor cell lines following photodynamic therapy with 5-aminolevulinic acid gave insight into the major molecular mechanisms induced by photodynamic therapy.

OBJECTIVE

The onset and progression of colorectal cancer (CRC) involves a cascade of genetic and/or epigenetic events. The aim of the present study was to address the DNA methylation status of genes relevant in colorectal carcinogenesis and its progression, such as genes frequently mutated in CRC, genes involved in the DNA repair and Wnt signaling pathway.

METHODS

We analyzed methylation status in totally 160 genes in 12 paired colorectal tumors and adjacent healthy mucosal tissues using the Illumina Infinium Human Methylation 450 BeadChip.

RESULTS

We found significantly aberrant methylation in 23 genes (NEIL1, NEIL3, DCLRE1C, NHEJ1, GTF2H5, CCNH, CTNNB1, DKK2, DKK3, FZD5 LRP5, TLE3, WNT2, WNT3A, WNT6, TCF7L1, CASP8, EDNRB1, GPC6, KIAA1804, MYO1B, SMAD2 and TTN). External validation by mRNA expression showed a good agreement between hypermethylation in cancer and down-regulated mRNA expression of the genes EDNRB1, GPC6 and SMAD2, and between hypomethylation and up-regulated mRNA expression of the CASP8 and DCLRE1C genes.

CONCLUSIONS

Aberrant methylation of the DCLRE1C and GPC6 genes are presented here for the first time and are therefore of special interest for further validation as novel candidate biomarker genes in CRC, and merit further validation with specific assays.