hPG\u3csub\u3e80\u3c/sub\u3e (Circulating Progastrin), a Novel Blood-Based Biomarker for Detection of Poorly Differentiated Neuroendocrine Carcinoma and Well Differentiated Neuroendocrine Tumors

Current blood-based biomarkers for neuroendocrine neoplasms (NENs) lack both sensitivity and specificity. Human circulating progastrin (hPG80) is a novel biomarker that can be easily measured in plasma by ELISA. This study is the first to examine hPG80 in NENs. Plasma hPG80 was quantified from 95 stage IV NEN patients, using DxPG80 technology (ECS Progastrin, Switzerland) and compared with hPG80 concentrations in two cohorts of healthy donor controls aged 50–80 (n = 252) and 18–25 (n = 137). Median hPG80 in NENs patients was 5.54 pM compared to 1.5 pM for the 50–80 controls and 0.29 pM the 18–25 cohort (p \u3c 0.0001). Subgroup analysis revealed median hPG80 levels significantly higher than for either control cohort in neuroendocrine carcinoma (NEC; n = 25) and neuroendocrine tumors (NET; n = 70) including the small-cell lung cancer (SCLC) sub-cohort (n = 13). Diagnostic accuracy, estimated by AUCs, was high for NENs, as well as both sub-groups (NEC/NET) when compared to the younger and older control groups. Plasma hPG80 in NENs may be a diagnostic blood biomarker for both low- and high-grade NENs; further study is warranted. A prospective multi-center trial is ongoing in NET to evaluate hPG80 as a means of monitoring disease (NCT04750954)

The type 1 diabetes gene TYK2 regulates beta-cell development and its responses to interferon-alpha

The TYK2 gene is associated with development of type 1 diabetes. Here the authors show that TYK2 regulates beta-cell development, but at the same time TYK2 inhibition in the islets prevents IFN alpha responses and enhances their survival against CD8(+) T-cell cytotoxicity; representing a potent therapeutic target to halt T1D progression. Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin producing pancreatic beta-cells. One of the genes associated with T1D is TYK2, which encodes a Janus kinase with critical roles in type-Iota interferon (IFN-Iota) mediated intracellular signalling. To study the role of TYK2 in beta-cell development and response to IFN alpha, we generated TYK2 knockout human iPSCs and directed them into the pancreatic endocrine lineage. Here we show that loss of TYK2 compromises the emergence of endocrine precursors by regulating KRAS expression, while mature stem cell-islets (SC-islets) function is not affected. In the SC-islets, the loss or inhibition of TYK2 prevents IFN alpha-induced antigen processing and presentation, including MHC Class Iota and Class Iota Iota expression, enhancing their survival against CD8(+) T-cell cytotoxicity. These results identify an unsuspected role for TYK2 in beta-cell development and support TYK2 inhibition in adult beta-cells as a potent therapeutic target to halt T1D progression.Peer reviewe

Fgfr3 Is a Transcriptional Target of Ap2δ and Ash2l-Containing Histone Methyltransferase Complexes

Polycomb (PcG) and trithorax (trxG) proteins play important roles in establishing lineage-specific genetic programs through induction of chromatin modifications that lead to gene silencing or activation. Previously, we described an association between the MLL/SET1 complexes and a highly restricted, gene-specific DNA-binding protein Ap2δ that is required for recruitment of the MLL/SET1 complex to target Hoxc8 specifically. Here, we reduced levels of Ap2δ and Ash2l in the neuroblastoma cell line, Neuro2A, and analyzed their gene expression profiles using whole-genome mouse cDNA microarrays. This analysis yielded 42 genes that are potentially co-regulated by Ap2δ and Ash2l, and we have identified evolutionarily conserved Ap2-binding sites in 20 of them. To determine whether some of these were direct targets of the Ap2δ-Ash2l complex, we analyzed several promoters for the presence of Ap2δ and Ash2l by chromatin immunoprecipitation (ChIP). Among the targets we screened, we identified Fgfr3 as a direct transcriptional target of the Ap2δ-Ash2l complex. Additionally, we found that Ap2δ is necessary for the recruitment of Ash2l-containing complexes to this promoter and that this recruitment leads to trimethylation of lysine 4 of histone H3 (H3K4me3). Thus, we have identified several candidate targets of complexes containing Ap2δ and Ash2l that can be used to further elucidate their roles during development and showed that Fgfr3 is a novel direct target of these complexes

Enhancer of zeste homolog 2 (EZH2) expression is an independent prognostic factor in renal cell carcinoma

Background: The enhancer of zeste homolog 2 (EZH2) gene exerts oncogene-like activities and its (over)expression has been linked to several human malignancies. Here, we studied a possible association between EZH2 expression and prognosis in patients with renal cell carcinoma (RCC). Methods: EZH2 protein expression in RCC specimens was analyzed by immunohistochemistry using a tissue microarray (TMA) containing RCC tumor tissue and corresponding normal tissue samples of 520 patients. For immunohistochemical assessment of EZH2 expression, nuclear staining quantity was evaluated using a semiquantitative score. The effect of EZH2 expression on cancer specific survival (CSS) was assessed by univariate and multivariate Cox regression analyses. Results: During follow-up, 147 patients (28%) had died of their disease, median follow-up of patients still alive was 6.0 years (range 0 - 16.1 years). EZH2 nuclear staining was present in tumor cores of 411 (79%) patients. A multivariate Cox regression analysis revealed that high nuclear EZH2 expression was an independent predictor of poor CSS (>25-50% vs. 0%: HR 2.72, p = 0.025) in patients suffering from non-metastatic RCC. Apart from high nuclear EZH2 expression, tumor stage and Fuhrman's grading emerged as significant prognostic markers. In metastatic disease, nuclear EZH2 expression and histopathological subtype were independent predictive parameters of poor CSS (EZH2: 1-5%: HR 2.63, p = 0.043, >5-25%: HR 3.35, p = 0.013, >25%-50%: HR 4.92, p = 0.003, all compared to 0%: HR 0.36, p = 0.025, respectively). Conclusions: This study defines EZH2 as a powerful independent unfavourable prognostic marker of CSS in patients with metastatic and non-metastatic RCC

Genome-wide DNA methylation analysis in cohesin mutant human cell lines

The cohesin complex has recently been shown to be a key regulator of eukaryotic gene expression, although the mechanisms by which it exerts its effects are poorly understood. We have undertaken a genome-wide analysis of DNA methylation in cohesin-deficient cell lines from probands with Cornelia de Lange syndrome (CdLS). Heterozygous mutations in NIPBL, SMC1A and SMC3 genes account for ∼65% of individuals with CdLS. SMC1A and SMC3 are subunits of the cohesin complex that controls sister chromatid cohesion, whereas NIPBL facilitates cohesin loading and unloading. We have examined the methylation status of 27 578 CpG dinucleotides in 72 CdLS and control samples. We have documented the DNA methylation pattern in human lymphoblastoid cell lines (LCLs) as well as identified specific differential DNA methylation in CdLS. Subgroups of CdLS probands and controls can be classified using selected CpG loci. The X chromosome was also found to have a unique DNA methylation pattern in CdLS. Cohesin preferentially binds to hypo-methylated DNA in control LCLs, whereas the differential DNA methylation alters cohesin binding in CdLS. Our results suggest that in addition to DNA methylation multiple mechanisms may be involved in transcriptional regulation in human cells and in the resultant gene misexpression in CdLS

The Role of EZH2 in the Regulation of the Activity of Matrix Metalloproteinases in Prostate Cancer Cells

Degradation of the extracellular matrix (ECM), a critical step in cancer metastasis, is determined by the balance between MMPs (matrix metalloproteinases) and their inhibitors TIMPs (tissue inhibitors of metalloproteinases). In cancer cells, this balance is shifted towards MMPs, promoting ECM degradation. Here, we show that EZH2 plays an active role in this process by repressing the expression of TIMP2 and TIMP3 in prostate cancer cells. The TIMP genes are derepressed by knockdown of EZH2 expression in human prostate cancer cells but repressed by overexpression of EZH2 in benign human prostate epithelial cells. EZH2 catalyzes H3K27 trimethylation and subsequent DNA methylation of the TIMP gene promoters. Overexpression of EZH2 confers an invasive phenotype on benign prostate epithelial cells; however, this phenotype is suppressed by cooverexpression of TIMP3. EZH2 knockdown markedly reduces the proteolytic activity of MMP-9, thereby decreasing the invasive activity of prostate cancer cells. These results suggest that the transcriptional repression of the TIMP genes by EZH2 may be a major mechanism to shift the MMPs/TIMPs balance in favor of MMP activity and thus to promote ECM degradation and subsequent invasion of prostate cancer cells

Remodeling of the chromatin structure of the facioscapulohumeral muscular dystrophy (FSHD) locus and upregulation of FSHD-related gene 1 (FRG1) expression during human myogenic differentiation

<p>Abstract</p> <p>Background</p> <p>Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder associated with the partial deletion of integral numbers of 3.3 kb D4Z4 DNA repeats within the subtelomere of chromosome 4q. A number of candidate FSHD genes, adenine nucleotide translocator 1 gene (<it>ANT1</it>), FSHD-related gene 1 (<it>FRG1</it>), <it>FRG2 </it>and <it>DUX4c</it>, upstream of the D4Z4 array (FSHD locus), and double homeobox chromosome 4 (<it>DUX4</it>) within the repeat itself, are upregulated in some patients, thus suggesting an underlying perturbation of the chromatin structure. Furthermore, a mouse model overexpressing <it>FRG1 </it>has been generated, displaying skeletal muscle defects.</p> <p>Results</p> <p>In the context of myogenic differentiation, we compared the chromatin structure and tridimensional interaction of the D4Z4 array and <it>FRG1 </it>gene promoter, and <it>FRG1 </it>expression, in control and FSHD cells. The <it>FRG1 </it>gene was prematurely expressed during FSHD myoblast differentiation, thus suggesting that the number of D4Z4 repeats in the array may affect the correct timing of <it>FRG1 </it>expression. Using chromosome conformation capture (3C) technology, we revealed that the <it>FRG1 </it>promoter and D4Z4 array physically interacted. Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the <it>FRG1</it>/4q-D4Z4 array loop in myotubes. The <it>FRG1 </it>promoter in both normal and FSHD myoblasts was characterized by H3K27 trimethylation and Polycomb repressor complex binding, but these repression signs were replaced by H3K4 trimethylation during differentiation. The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation.</p> <p>Conclusion</p> <p>We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of <it>in cis </it>chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.</p

Polycomb CBX7 Directly Controls Trimethylation of Histone H3 at Lysine 9 at the p16 Locus

BACKGROUND: H3K9 trimethylation (H3K9me3) and binding of PcG repressor complex-1 (PRC1) may play crucial roles in the epigenetic silencing of the p16 gene. However, the mechanism of the initiation of this trimethylation is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we found that upregulating the expression of PRC1 component Cbx7 in gastric cancer cell lines MGC803 and BGC823 led to significantly suppress the expression of genes within the p16-Arf-p15 locus. H3K9me3 formation was observed at the p16 promoter and Regulatory Domain (RD). CBX7 and SUV39H2 binding to these regions were also detectable in the CBX7-stably upregulated cells. CBX7-SUV39H2 complexes were observed within nucleus in bimolecular fluorescence complementation assay (BiFC). Mutations of the chromodomain or deletion of Pc-box abolished the CBX7-binding and H3K9me3 formation, and thus partially repressed the function of CBX7. SiRNA-knockdown of Suv39h2 blocked the repressive effect of CBX7 on p16 transcription. Moreover, we found that expression of CBX7 in gastric carcinoma tissues with p16 methylation was significantly lower than that in their corresponding normal tissues, which showed a negative correlation with transcription of p16 in gastric mucosa. CONCLUSION/SIGNIFICANCE: These results demonstrated for the first time, to our knowledge, that CBX7 could initiate H3K9me3 formation at the p16 promoter

Antagonism between DNA and H3K27 Methylation at the Imprinted Rasgrf1 Locus

At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development

FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer

FOXA1 expression correlates with the breast cancer luminal subtype and patient survival. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with the downregulation of FOXA1 expression. Knockdown of BRCA1 resulted in the downregulation of FOXA1 expression and enhancement of FOXA1 promoter methylation in MCF-7 breast cancer cells, whereas the reconstitution of BRCA1 in Brca1-deficent mouse mammary epithelial cells (MMECs) promoted Foxa1 expression and methylation. These data suggest that BRCA1 suppresses FOXA1 hypermethylation and silencing. Consistently, the treatment of MMECs with the DNA methylation inhibitor 5-aza-2'-deoxycitydine induced Foxa1 mRNA expression. Furthermore, treatment with GSK126, an inhibitor of EZH2 methyltransferase activity, induced FOXA1 expression in BRCA1-deficient but not in BRCA1-reconstituted MMECs. Likewise, the depletion of EZH2 by small interfering RNA enhanced FOXA1 mRNA expression. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNA methyltransferases (DNMT)1/3a/3b and H3K27me3 are recruited to the endogenous FOXA1 promoter, further supporting the hypothesis that these proteins interact to modulate FOXA1 methylation and repression. Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity. We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter. These associations were validated in a familial breast cancer patient cohort. Integrated analysis of the global gene methylation and expression profiles of a set of 33 familial breast tumours revealed that FOXA1 promoter methylation is inversely correlated with the transcriptional expression of FOXA1 and that BRCA1 mutation breast cancer is significantly associated with FOXA1 methylation and downregulation of FOXA1 expression, providing physiological evidence to our findings that FOXA1 expression is regulated by methylation and chromatin silencing and that BRCA1 maintains FOXA1 expression through suppressing FOXA1 gene methylation in breast cancer.Oncogene advance online publication, 22 December 2014; doi:10.1038/onc.2014.421.published_or_final_versio