A four-gene methylation marker panel as triage test in high-risk human papillomavirus positive patients.

Cervical neoplasia-specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population-based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia-specific DNA methylation markers and to design and validate a methylation marker panel for triage of high-risk human papillomavirus (hr-HPV) positive patients. First, high-throughput quantitative methylation-specific PCRs (QMSP) on a novel OpenArray™ platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCycler® MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four-gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN3+) and 65% (44/68) CIN2+ were detected, with 21% positive cases for ≤CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr-HPV testing combined with our four-gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr-HPV testing in combination with conventional cytology. In conclusion, our four-gene methylation panel might provide an alternative triage test after primary hr-HPV testing.

E2Fs mediate a fundamental cell-cycle deregulation in high-grade serous ovarian carcinomas.

Several studies described a role for the E2F/Rb pathway in ovarian serous carcinomas (SCAs). Since E2F/Rb pathway deregulation is a general hallmark of human cancer, it remains unclear whether this deregulation is of particular importance in SCAs or whether it reflects a common oncological feature. Here, we have clarified this issue by the examination of microarray expression profiles of SCAs and particularly by the comparison with another, less malignant, ovarian cancer type, serous borderline tumours (SBTs). Results were validated by quantitative RT-PCR, both on the microarray samples and on an independent panel, and TP53 mutation analysis was performed. This integrated analysis revealed a significant increase in the expression of the transcription factors E2F1 and E2F3 in SCAs, when compared to SBTs. This was associated with vast overexpression of E2F target genes in SCAs compared to SBTs. High-grade SCAs in particular exhibited a major deregulated E2F target expression pattern. Generally, overexpression of E2F targets in SCAs appeared to be well structured since those targets considered negative regulators of the cell cycle or promoters of apoptosis were usually not overexpressed in SCAs. Similar to E2F target deregulation, TP53 mutations were identified in SCA3s, to a lesser extent in SCA1s, and not in SBTs. These results suggest that a structured, generally up-regulated E2F transcription factor activity is associated with a global cell-cycle disturbance in high-grade SCAs and exceeds typical E2F/Rb pathway disruption in tumours, at least compared with SBTs.

Multiple putative oncogenes at the chromosome 20q amplicon contribute to colorectal adenoma to carcinoma progression.

OBJECTIVE: This study aimed to identify the oncogenes at 20q involved in colorectal adenoma to carcinoma progression by measuring the effect of 20q gain on mRNA expression of genes in this amplicon. METHODS: Segmentation of DNA copy number changes on 20q was performed by array CGH (comparative genomic hybridisation) in 34 non-progressed colorectal adenomas, 41 progressed adenomas (ie, adenomas that present a focus of cancer) and 33 adenocarcinomas. Moreover, a robust analysis of altered expression of genes in these segments was performed by microarray analysis in 37 adenomas and 31 adenocarcinomas. Protein expression was evaluated by immunohistochemistry on tissue microarrays. RESULTS: The genes C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5, mapping at 20q, were significantly overexpressed in carcinomas compared with adenomas as a consequence of copy number gain of 20q. CONCLUSION: This approach revealed C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5 genes to be important in chromosomal instability-related adenoma to carcinoma progression. These genes therefore may serve as highly specific biomarkers for colorectal cancer with potential clinical applications.

The zinc finger protein A20 inhibits TNF-induced NF-kappaB-dependent gene expression by interfering with an RIP- or TRAF2-mediated transactivation signal and directly binds to a novel NF-kappaB-inhibiting protein ABIN.

The zinc finger protein A20 is a tumor necrosis factor (TNF)- and interleukin 1 (IL-1)-inducible protein that negatively regulates nuclear factor-kappa B (NF-kappaB)-dependent gene expression. However, the molecular mechanism by which A20 exerts this effect is still unclear. We show that A20 does not inhibit TNF- induced nuclear translocation and DNA binding of NF-kappaB, although it completely prevents the TNF- induced activation of an NF-kappaB-dependent reporter gene, as well as TNF-induced IL-6 and granulocyte macrophage-colony stimulating factor gene expression. Moreover, NF-kappaB activation induced by overexpression of the TNF receptor-associated proteins TNF receptor-associated death domain protein (TRADD), receptor interacting protein (RIP), and TNF recep- tor-associated factor 2 (TRAF2) was also inhibited by expression of A20, whereas NF-kappaB activation induced by overexpression of NF-kappaB-inducing kinase (NIK) or the human T cell leukemia virus type 1 (HTLV-1) Tax was unaffected. These results demonstrate that A20 inhibits NF-kappaB-dependent gene expression by interfering with a novel TNF-induced and RIP- or TRAF2-mediated pathway that is different from the NIK-IkappaB kinase pathway and that is specifically involved in the transactivation of NF-kappaB. Via yeast two-hybrid screening, we found that A20 binds to a novel protein, ABIN, which mimics the NF-kappaB inhibiting effects of A20 upon overexpression, suggesting that the effect of A20 is mediated by its interaction with this NF-kappaB inhibiting protein, ABIN.

The human homologue of Caenorhabditis elegans CED-6 specifically promotes phagocytosis of apoptotic cells.

A key feature of the process of programmed cell death (apoptosis) is the efficiency with which the dying cells are recognized and engulfed by phagocytes [1]. Apoptotic cells are rapidly cleared either by neighbouring cells acting as semi-professional phagocytes or by experts of the macrophage line, so that an inflammatory response is avoided [2]. The Caenorhabditis elegans gene ced-6 is required for efficient engulfment of apoptotic cells [3] and is one of a group of genes that define two partially redundant parallel pathways for the engulfment process [4] [5]. These pathways may be conserved across evolution, as two other engulfment genes have human homologues. A CED-5 homologue is part of a human CrkII-DOCK180-Rac signaling pathway proposed to mediate cytoskeletal reorganization [6] [7] [8] and a CED-7 homologue is similar to the ABC transporters [9] [10]. Here, we report the cloning and characterization of human CED-6, a human homologue of C. elegans CED-6. The 34 kDa hCED-6 protein is expressed in most tissues, some human cancer cells, and in primary human macrophages. We developed an assay that quantitates the phagocytic activity of mammalian macrophages: the number of apoptotic cells that have been internalized is measured by the uptake of lacZ-positive apoptotic cells by adherent transgenic macrophages. The results of this assay demonstrate that overexpression of hCED-6 promotes phagocytosis only of apoptotic cells and suggest that hCED-6 is the mammalian orthologue of C. elegans CED-6 and is a part of a highly conserved pathway that specifically mediates the phagocytosis of apoptotic cells.

RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis.

Oral and oropharyngeal squamous cell carcinoma (OOSCC) have a low survival rate, mainly due to metastasis to the regional lymph nodes. For optimal treatment of these metastases, a neck dissection is required; however, inaccurate detection methods results in under- and over-treatment. New DNA prognostic methylation biomarkers might improve lymph node metastases detection. To identify epigenetically regulated genes associated with lymph node metastases, genome-wide methylation analysis was performed on 6 OOSCC with (pN+) and 6 OOSCC without (pN0) lymph node metastases and combined with a gene expression signature predictive for pN+ status in OOSCC. Selected genes were validated using an independent OOSCC cohort by immunohistochemistry and pyrosequencing, and on data retrieved from The Cancer Genome Atlas. A two-step statistical selection of differentially methylated sequences revealed 14 genes with increased methylation status and mRNA downregulation in pN+ OOSCC. RAB25, a known tumor suppressor gene, was the highest-ranking gene in the discovery set. In the validation sets, both RAB25 mRNA (P = 0.015) and protein levels (P = 0.012) were lower in pN+ OOSCC. RAB25 mRNA levels were negatively correlated with RAB25 methylation levels (P < 0.001) but RAB25 protein expression was not. Our data revealed that promoter methylation is a mechanism resulting in downregulation of RAB25 expression in pN+ OOSCC and decreased expression is associated with lymph node metastasis. Detection of RAB25 methylation might contribute to lymph node metastasis diagnosis and serve as a potential new therapeutic target in OOSCC.

Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3.

BACKGROUND: Assessment of DNA promoter methylation markers in cervical scrapings for the detection of cervical intraepithelial neoplasia (CIN) and cervical cancer is feasible, but finding methylation markers with both high sensitivity as well as high specificity remains a challenge. In this study, we aimed to identify new methylation markers for the detection of high-grade CIN (CIN2/3 or worse, CIN2+) by using innovative genome-wide methylation analysis (MethylCap-seq). We focused on diagnostic performance of methylation markers with high sensitivity and high specificity considering any methylation level as positive. RESULTS: MethylCap-seq of normal cervices and CIN2/3 revealed 176 differentially methylated regions (DMRs) comprising 164 genes. After verification and validation of the 15 best discriminating genes with methylation-specific PCR (MSP), 9 genes showed significant differential methylation in an independent cohort of normal cervices versus CIN2/3 lesions (p < 0.05). For further diagnostic evaluation, these 9 markers were tested with quantitative MSP (QMSP) in cervical scrapings from 2 cohorts: (1) cervical carcinoma versus healthy controls and (2) patients referred from population-based screening with an abnormal Pap smear in whom also HPV status was determined. Methylation levels of 8/9 genes were significantly higher in carcinoma compared to normal scrapings. For all 8 genes, methylation levels increased with the severity of the underlying histological lesion in scrapings from patients referred with an abnormal Pap smear. In addition, the diagnostic performance was investigated, using these 8 new genes and 4 genes (previously identified by our group: C13ORF18, JAM3, EPB41L3, and TERT). In a triage setting (after a positive Pap smear), sensitivity for CIN2+ of the best combination of genes (C13ORF18/JAM3/ANKRD18CP) (74 %) was comparable to hrHPV testing (79 %), while specificity was significantly higher (76 % versus 42 %, p ≤ 0.05). In addition, in hrHPV-positive scrapings, sensitivity and specificity for CIN2+ of this best-performing combination was comparable to the population referred with abnormal Pap smear. CONCLUSIONS: We identified new CIN2/3-specific methylation markers using genome-wide DNA methylation analysis. The diagnostic performance of our new methylation panel shows higher specificity, which should result in prevention of unnecessary colposcopies for women referred with abnormal cytology. In addition, these newly found markers might be applied as a triage test in hrHPV-positive women from population-based screening. The next step before implementation in primary screening programs will be validation in population-based cohorts.

Identification of a new caspase homologue: caspase-14.

Caspases are cysteinyl aspartate-specific proteinases, many of which play a central role in apoptosis. Here, we report the identification of a new murine caspase homologue, viz. caspase-14. It is most related to human/murine caspase-2 and human caspase-9, possesses all the typical amino acid residues of the caspases involved in catalysis, including the QACRG box, and contains no or only a very short prodomain. Murine caspase-14 shows 83% similarity to human caspase-14. Human caspase-14 is assigned to chromosome 19p13.1. Northern blot analysis revealed that mRNA expression of caspase-14 is undetectable in all mouse adult tissues examined except for skin, while it is abundantly expressed in mouse embryos. In contrast to many other caspase family members, murine caspase-14 is not cleaved by granzyme B, caspase-1, caspase-2, caspase-3, caspase-6, caspase-7 or caspase-11, but is weakly processed into p18 and p11 subunits by murine caspase-8. No aspartase activity of murine caspase-14 could be generated by bacterial or yeast expression. Transient overexpression of murine caspase-14 in mammalian cells did not elicit cell death and did not interfere with caspase-8-induced apoptosis. In conclusion, caspase-14 is a member of the caspase family but no proteolytic or biological activities have been identified so far. The high constitutive expression levels in embryos and specific expression in adult skin suggest a role in ontogenesis and skin physiology.

A20, an inhibitor of cell death, self-associates by its zinc finger domain.

A20 is a primary response gene which is induced after monocyte adherence or cytokine stimulation of a variety of cells. The A20 protein belongs to a novel class of Cys2/Cys2 zinc finger proteins, and has been characterized as an inhibitor of both apoptotic and necrotic cell death. In order to clarify its molecular mechanism of action, we used the yeast-based two-hybrid system to screen for A20-associated proteins. Here we report that A20 is able to self-associate, and demonstrate that the latter interaction is mediated by its zinc finger domain.

Characterization of seven murine caspase family members.

Seven members of the murine caspase (mCASP) family were cloned and functionally characterized by transient overexpression: mCASP-1 (mICE), mCASP-2 (Ich1), mCASP-3 (CPP32), mCASP-6 (Mch2), mCASP-7 (Mch3), mCASP-11 (TX) and mCASP-12. mCASP-11 is presumably the murine homolog of human CASP-4. Although mCASP-12 is related to human CASP-5 (ICErel-III), it is most probably a new CASP-1 family member. On the basis of sequence homology, the caspases can be divided into three subfamilies: first, mCASP-1, mCASP-11 and mCASP-12; second, mCASP-2; third, mCASP-3, mCASP-6 and mCASP-7. The tissue distribution of the CASP-1 subfamily transcripts is more restricted than that of the CASP-3 subfamily transcripts, suggesting that the transcriptional regulation of the CASP members within one subfamily is related, but is quite different between the CASP-1 and the CASP-3 subfamilies. Transient overexpression of each of the seven CASPs induced apoptosis in mammalian cells. Only two, mCASP-1 as well as mCASP-3, were able to process precursor interleukin (IL)-1beta to biologically active IL-1beta. In addition, mCASP-3 is the predominant PARP-cleaving enzyme in vivo.

Identification of caspases that cleave presenilin-1 and presenilin-2. Five presenilin-1 (PS1) mutations do not alter the sensitivity of PS1 to caspases.

Mutations in the presenilin (PS) genes PSI and PS2 are involved in Alzheimer's disease (AD). Recently, apoptosis-associated cleavage of PS proteins was identified. Here we demonstrate that PS1 as well as PS2 are substrates for different members of the caspase protein family. Remarkably, the caspases acting on PS1 could be subdivided in two groups. One group, containing caspase-8, -6 and -11, cleaved PSI after residues ENDD329 and to a lesser extent after residues AQRD341. A second group consisting of caspase-3, -7 and -1 acted uniquely on AQRD341. Importantly, these two cleavage sites were also recognized by caspases in the C-terminal PS1 fragment produced by constitutive proteolysis. In decreasing order of activity, caspase-8, -3, -1, -6 and -7 proteolysed PS2 at the recognition site D326SYD329. Caspase-8 and -3 exhibited the highest proteolytic activity on both PS1 and PS2. PS1 and PS2 were not hydrolyzed by caspase-2 and PS2 also not by caspase-11. None of five missense mutations affected the sensitivity of PSI to caspase-mediated cleavage. This suggests that AD pathogenesis associated with PS1 missense mutations cannot be explained by a change in caspase-dependent processing.

TNF-induced intracellular signaling leading to gene induction or to cytotoxicity by necrosis or by apoptosis.

TNF-induced apoptosis, e.g. in murine PC60 cells, requires the TNF receptor p55 (TNF-R55) and the TNF receptor p75 (TNF-R75); the latter even does not have to be triggered. The intracellular domain of TNF-R55 can be activated in the cytosol by linking it to the trimeric CAT protein; induction of this fusion protein leads to a full TNF response. A new MAP kinase, p38, has been shown to be also activated by TNF. This activation is essential for gene induction, but not for cytotoxicity in L929 cells. TNF treatment of L929 leads to reactive oxygen formation in the mitochondria, resulting in cell death by necrosis. TNF treatment of many other cell types results in apoptosis, and this process involves activation of one or more ICE homologs (IHO). In the mouse, seven cysteine proteases of the IHO family have been cloned and partially characterized. One or more of these IHOs is involved in cell killing by proteolysis of critical substrate(s). One substrate, which may be a key effector molecule in the apoptotic process, is PITSLRE kinase.

Expression of type 2 orexin receptor in human endometrium and its epigenetic silencing in endometrial cancer.

CONTEXT: Orexins A and B are neuropeptides that bind and activate 2 types of receptors. In addition to direct action in the brain, the orexinergic system has broader implications in peripheral organs, and it has been proposed to have a role in the induction of apoptosis. There are very few data on the endometrium. OBJECTIVE: The expression and epigenetic regulation of type 2 orexin receptor (OX2R) was investigated in the human endometrium as well as in endometrial endometrioid carcinoma (EEC). METHODS: OX2R localization was studied by immunohistochemistry in normal endometrium (n = 24) and in EEC (n = 32). The DNA methylation status of a CpG island located in the first exon of OX2R was analyzed by bisulfite sequencing in normal (n = 18), EEC (n = 34), and 3 endometrial cell lines. On the latter, mRNA expression and Western blotting as well as in vitro induction with orexin were performed. RESULTS: Expression of the OX2R protein was detected in normal endometrial epithelia, whereas it was frequently lacking in EEC. This loss was associated with hypermethylation of OX2R in EEC in comparison with normal endometrium (median CpG methylation percentages of 48.85% and 5.85%, respectively). In cell lines, hypermethylation correlated with weak OX2R expression. Additionally, in vitro treatment of the 3 EEC cell lines with orexins A and B did not result in proliferation change CONCLUSIONS: Altogether our data provide evidence for the epigenetic silencing of OX2R in EEC. The implication of the OX2R loss in tumoral progression remains to be elucidated.

A20 inhibits NF-kappaB activation independently of binding to 14-3-3 proteins.

The A20 protein, which belongs to a class of Cys2/Cys2 zinc finger proteins, has been characterized as an inhibitor of NF-kappaB activation. In order to clarify its molecular mechanism of action, the yeast two-hybrid system was used to screen for interacting proteins. We report that different isoforms of 14-3-3 proteins, viz. eta and zeta, are able to bind A20, involving the 14-3-3-binding motif RSKSDP located between zinc fingers 3 and 4. However, A20 mutants that no longer associated with 14-3-3 proteins could still fully inhibit NF-kappaB activation induced by tumor necrosis factor, interleukin-1beta or phorbol 12-myristate 13-acetate, thus excluding a crucial role for 14-3-3 interaction in this A20 function.

Functional characterization of the prodomain of interleukin-1beta-converting enzyme.

Interleukin-1beta-converting enzyme (ICE) has been identified as the main protease responsible for maturation of the prodomain of interleukin-1beta. Recently, it was shown to belong to a larger gene family, members of which play an important role in programmed cell death. A common feature of the ICE family proteases is the presence of a prodomain that has been hypothesized to keep the enzyme in an inactive form. Expression analysis in yeast revealed autocatalytic degradation of p45ICE, but not of p30ICE lacking a prodomain. We further demonstrate that p45ICE, in which the critical cysteine has been mutated, is still able to dimerize in vivo. Dimerization requires the prodomain and occurs prior to autoprocessing. These results provide evidence for a regulatory role of the prodomain of ICE.

GAL4 is a substrate for caspases: implications for two-hybrid screening and other GAL4-based assays.

Yeast two-hybrid technology as well as mammalian reporter assays use fusions between a protein of interest and the GAL4 DNA-binding domain (GAL4DB). We demonstrate that expression of a GAL4DB/caspase-1 chimeric protein in yeast leads to autoproteolytic cleavage of GAL4DB. Moreover, recombinant GAL4DB is a good in vitro substrate for recombinant caspase-1 and several other caspases. Cleavage sites map at the C-terminus of GAL4DB and result in release of the fused protein. The finding that GAL4DB can be cleaved by caspases has important implications for the use of caspases in two-hybrid analysis and in the interpretation of mammalian assays based on GAL4-dependent reporter gene expression.