Characterizing caspase-1 involvement during esophageal disease progression.

Barrett's esophagus (BE) is an inflammatory condition and a neoplastic precursor to esophageal adenocarcinoma (EAC). Inflammasome signaling, which contributes to acute and chronic inflammation, results in caspase-1 activation leading to the secretion of IL-1ß and IL-18, and inflammatory cell death (pyroptosis). This study aimed to characterize caspase-1 expression, and its functional importance, during disease progression to BE and EAC. Three models of disease progression (Normal-BE-EAC) were employed to profile caspase-1 expression: (1) a human esophageal cell line model; (2) a murine model of BE; and (3) resected tissue from BE-associated EAC patients. BE patient biopsies and murine BE organoids were cultured ex vivo in the presence of a caspase-1 inhibitor, to determine the importance of caspase-1 for inflammatory cytokine and chemokine secretion.Epithelial caspase-1 expression levels were significantly enhanced in BE (p < 0.01). In contrast, stromal caspase-1 levels correlated with histological inflammation scores during disease progression (p < 0.05). Elevated secretion of IL-1ß from BE explanted tissue, compared to adjacent normal tissue (p < 0.01), confirmed enhanced activity of caspase-1 in BE tissue. Caspase-1 inhibition in LPS-stimulated murine BE organoids caused a significant reduction in IL-1ß (p < 0.01) and CXCL1 (p < 0.05) secretion, confirming the importance of caspase-1 in the production of cytokines and chemokines associated with disease progression from BE to EAC. Targeting caspase-1 activity in BE patients should therefore be tested as a novel strategy to prevent inflammatory complications associated with disease progression.

HLA-DR expression in tumor epithelium is an independent prognostic indicator in esophageal adenocarcinoma patients.

Esophageal adenocarcinoma (EAC) is an aggressive cancer with poor prognosis, and incidence is increasing rapidly in the Western world. Measurement of immune markers has been shown to have prognostic significance in a growing number of cancers, but whether this is true for EAC has yet to be evaluated. This study aimed to characterize HLA-DR expression in the esophagus across the inflammation to cancer progression sequence and to assess the prognostic significance of HLA-DR expression in EAC. Tissue microarrays (TMA) were constructed from esophageal tissue taken from patients at different stages in the cancer progression sequence; normal, esophagitis, Barrett's esophagus (BE), low- and high-grade dysplasia (LGD, HGD) and EAC. HLA-DR expression in tissue epithelium and stroma was assessed by immunohistochemistry. HLA-DR expression increased early in the inflammation to cancer progression sequence; with higher expression detected in esophagitis and BE compared to normal tissue. Patients with low (<50%) HLA-DR expression in the EAC tumor epithelium had significantly worse survival outcomes, compared to those with high expression, in both the tumor core (hazard ratio, HR = 2.178, p = 0.024, n = 70) and leading edge (HR = 2.86, p = 0.013, n = 41). Multivariate analysis demonstrated that low HLA-DR expression in leading edge tumor epithelium was an independent predictor of poor survival, associated with a 2.8-fold increase in disease-associated death (p = 0.023). This study shows that HLA-DR is an independent prognostic marker in EAC tumor epithelium. This may have implications for patient stratification strategies as well as EAC tumor immunology.

Hypoxia-induced epigenetic modifications are associated with cardiac tissue fibrosis and the development of a myofibroblast-like phenotype.

Ischemia caused by coronary artery disease and myocardial infarction leads to aberrant ventricular remodeling and cardiac fibrosis. This occurs partly through accumulation of gene expression changes in resident fibroblasts, resulting in an overactive fibrotic phenotype. Long-term adaptation to a hypoxic insult is likely to require significant modification of chromatin structure in order to maintain the fibrotic phenotype. Epigenetic changes may play an important role in modulating hypoxia-induced fibrosis within the heart. Therefore, the aim of the study was to investigate the potential pro-fibrotic impact of hypoxia on cardiac fibroblasts and determine whether alterations in DNA methylation could play a role in this process. This study found that within human cardiac tissue, the degree of hypoxia was associated with increased expression of collagen 1 and alpha-smooth muscle actin (ASMA). In addition, human cardiac fibroblast cells exposed to prolonged 1% hypoxia resulted in a pro-fibrotic state. These hypoxia-induced pro-fibrotic changes were associated with global DNA hypermethylation and increased expression of the DNA methyltransferase (DNMT) enzymes DNMT1 and DNMT3B. Expression of these methylating enzymes was shown to be regulated by hypoxia-inducible factor (HIF)-1α. Using siRNA to block DNMT3B expression significantly reduced collagen 1 and ASMA expression. In addition, application of the DNMT inhibitor 5-aza-2'-deoxycytidine suppressed the pro-fibrotic effects of TGFß. Epigenetic modifications and changes in the epigenetic machinery identified in cardiac fibroblasts during prolonged hypoxia may contribute to the pro-fibrotic nature of the ischemic milieu. Targeting up-regulated expression of DNMTs in ischemic heart disease may prove to be a valuable therapeutic approach.

miRNA profiles as a predictor of chemoresponsiveness in Wilms' tumor blastema.

The current SIOP treatment protocol for Wilms' tumor involves pre-operative chemotherapy followed by nephrectomy. Not all patients benefit equally from such chemotherapy. The aim of this study was to generate a miRNA profile of chemo resistant blastemal cells in high risk Wilms' tumors which might serve as predictive markers of therapeutic response at the pre-treatment biopsy stage. We have shown here that unsupervised hierarchical clustering of genome-wide miRNA expression profiles can clearly separate intermediate risk tumors from high risk tumors. A total of 29 miRNAs were significantly differentially expressed between post-treatment intermediate risk and high risk groups, including miRNAs that have been previously linked to chemo resistance in other cancer types. Furthermore, 7 of these 29 miRNAs were already at the pre-treatment biopsy stage differentially expressed between cases ultimately deemed intermediate risk compared to high risk. These miRNA alterations include down-regulation in high risk cases of miR-193a.5p, miR-27a and the up-regulation of miR-483.5p, miR-628.5p, miR-590.5p, miR-302a and miR-367. The demonstration of such miRNA markers at the pre-treatment biopsy stage could permit stratification of patients to more tailored treatment regimens.

5-AZA-2'-deoxycytidine induced demethylation influences N-glycosylation of secreted glycoproteins in ovarian cancer.

Glycosylation is the most common posttranslational modification of proteins and is highly reflective of changes in the environment of a cell. Epigenetic modifications to the genome are stably transmitted to daughter cells without the requirement for genetic sequence alterations. Aberrant regulation of both epigenetic programming and glycosylation patterning are integral aspects of carcinogenesis. The objective of this study was to determine the interplay between these two complex cellular processes. We demonstrate that global DNA methylation changes in ovarian cancer epithelial cells (OVCAR3) resulted in significant alterations in the glycosylation of secreted glycoproteins. These changes included a reduction in core fucosylation, increased branching and increased sialylation. We further show that the change in core fucose levels was mirrored by altered expression of GMDS and FX, key enzymes in fucose biosynthesis. Alterations in the expression of key glycosyltransferase enzymes such as MGAT5 reflect the changes seen in the branching and sialylation of secreted glycans. Overall, our results highlight that modifications to the epigenetic machinery have a profound effect on the glycan structures generated by cells, which may be a key step in understanding metastasis and drug resistance during cancer progression.

MAD2 downregulation in hypoxia is independent of promoter hypermethylation.

Aberrant expression of the MAD2 protein has been linked to chromosomal instability, malignant transformation and chemoresistance. Although reduced MAD2 expression is well recognised in human cancer cell lines, the mechanism(s) underlying its downregulation remain elusive. The objective of this study was to establish the impact of hypoxia on MAD2 expression and to investigate the potential role of aberrant promoter methylation as a possible mechanism of MAD2 downregulation. For this purpose, three ovarian cancer cell lines, displaying differing levels of MAD2, were treated with chromatin modifying drugs, pre and post-hypoxia exposure and a DHPLC analysis of DNA promoter methylation carried out. We show that hypoxia induces downregulation of MAD2 expression, independently of MAD2 promoter methylation. We also show no evidence of MAD2 promoter methylation in breast and prostate cancer cells or in breast cancer clinical material. While our findings provide no evidence for MAD2 promoter methylation, we show a concomitant upregulation of p21 with downregulation of MAD2 in hypoxia. Our in vitro results were also confirmed in an ovarian cancer tissue microarray (TMA), where a reciprocal staining of MAD2 and CAIX was found in 21/60 (35%) of tumours. In summary, MAD2 downregulation may be a crucial mechanism by which hypoxic cells become chemorefractory. This stems from our previous work where we demonstrated that MAD2 downregulation induces cellular senescence, a viable cellular fate, with resultant cellular resistance to paclitaxel. Moreover, MAD2 downregulation could play a central role in the induction of chemoresistance in hypoxia, a key tumour microenvironment associated with chemoresistance.

Epigenetics, the epicenter of the hypoxic response.

It is becoming increasingly apparent that epigenetics plays a crucial role in the cellular response to hypoxia. Such epigenetic regulation may work hand in hand with the hypoxia-induced transcription factor (HIF) family or may contribute in a more substantial way to the maintenance of a hypoxia-adapted cellular phenotype long after HIF has initiated the immediate response pathways. In this article we discuss the current research implicating epigenetic mechanisms in the cellular response to hypoxic environments. This includes; the role of epigenetics in both the stabilization and binding of HIF to its transcriptional targets, the role of histone demethylase enzymes following direct HIF transactivation, and finally, the impact of hypoxic environments on global patterns of histone modifications and DNA methylation.

Hyperacetylation in prostate cancer induces cell cycle aberrations, chromatin reorganization and altered gene expression profiles.

Histone acetylation is a fundamental mechanism in the regulation of local chromatin conformation and gene expression. Research has focused on the impact of altered epigenetic environments on the expression of specific genes and their pathways. However, changes in histone acetylation also have a global impact on the cell. In this study we used digital texture analysis to assess global chromatin patterns following treatment with trichostatin A (TSA) and have observed significant alterations in the condensation and distribution of higher-order chromatin, which were associated with altered gene expression profiles in both immortalised normal PNT1A prostate cell line and androgen-dependent prostate cancer cell line LNCaP. Furthermore, the extent of TSA-induced disruption was both cell cycle and cell line dependent. This was illustrated by the identification of sub-populations of prostate cancer cells expressing high levels of H3K9 acetylation in the G(2)/M phase of the cell cycle that were absent in normal cell populations. In addition, the analysis of enriched populations of G(1) cells showed a global decondensation of chromatin exclusively in normal cells.

Generation of an epigenetic signature by chronic hypoxia in prostate cells.

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.

Urinary insulin-like growth factor 2 identifies the presence of urothelial carcinoma of the bladder.

OBJECTIVE: To examine urinary insulin-like growth factor 2 (IGF-2) levels in patient urine samples and determine the potential of IGF-2 as a marker for the presence of urothelial carcinoma of the bladder (UCB). PATIENTS AND METHODS: The current gold standard for diagnosis of UCB is cystoscopy and cytological analysis. The identification of an accurate urine marker for UCB with the potential to replace unnecessary cystoscopy would benefit patients with UCB and others investigated after detecting haematuria. In the present study, we analysed 65 urine samples, and optimized an enzyme-linked immunosorbent assay-based approach to measure urinary levels of IGF-2. RESULTS: Based on a threshold of 5.4 ng/mL, patients with UCB have significantly elevated levels of urinary IGF-2 (P = 0.009) and this difference remained significant after adjustment for age and sex (P = 0.04). Sensitivity and specificity values of 80% and 52%, respectively, were determined for urinary IGF-2 alone and when combined with nuclear matrix protein 22 (NMP22; an approved biomarker for detection of UCB). There was a positive correlation between urinary IGF-2 levels and NMP22 levels in patient urine samples and the combined assay improved the detection of UCB (sensitivity 85% and specificity 52%). CONCLUSION: Substantiated evidence has identified IGF-2 as a valuable marker for UCB. In addition, the novel observations of the present study have shown that aberrant levels of IGF-2 occurring in the presence of UCB, can now be determined through a simple and inexpensive urine assay.

Recurrence of urothelial carcinoma of the bladder: a role for insulin-like growth factor-II loss of imprinting and cytoplasmic E-cadherin immunolocalization.

PURPOSE: This study documents the frequency of insulin-like growth factor-II (IGF-II) loss of imprinting (LOI) in a series of 87 bladder tissues. E-cadherin (CDH1) immunolocalization was also investigated due to the known redistribution of this adherence protein to the cytoplasm following exogenous exposure to IGF-II. EXPERIMENTAL DESIGN: Informative IGF-II cases were identified following DNA-PCR amplification and subsequent sequencing of the transcribable ApaI RFLP in exon 9 of IGF-II. Similar approaches using primer-specific cDNA templates identified the imprinting status of IGF-II in these informative cases. CDH1 cellular localization was assessed on a tissue microarray platform of 114 urothelial carcinoma of the bladder (UCB) cases (70 pT(a) noninvasive and 44 pT(1) lamina propria invasive) using the commercially available Novocastra antibody. RESULTS: IGF-II LOI was evident in 7 of 17 (41%) UCB tumors and 4 of 11 (36%) tumor-associated normal urothelial samples. Two of four pT(1) grade 3 tumors, the subject of much debate concerning their suitability for radical cystectomy, showed LOI at the IGF-II locus. In those tumors showing IGF-II LOI, 4 of 7 (57%) displayed concomitant CDH1 cytoplasmic staining. In contrast, only 3 of 10 (30%) IGF-II maintenance of imprinting tumors had concomitant CDH1 cytoplasmic localization. UCB cell lines displaying cytoplasmic CDH1 immunolocalization expressed significantly higher levels of IGF-II (CAL29, HT1376, and RT112) compared with RT4, a cell line displaying crisp membranous CDH1 staining. Finally, cytoplasmic CDH1 staining was an independent predictor of a shorter time to recurrence independent of tumor grade and stage. CONCLUSIONS: We suggest that CDH1 cytoplasmic immunolocalization as a result of increased IGF-II levels identifies those nonmuscle invasive presentations most likely to recur and therefore might benefit from more radical nonconserving bladder surgery.

NXY-059 maintains Akt activation and inhibits release of cytochrome C after focal cerebral ischemia.

Stroke is the third leading cause of death in the US, with a prevalence of 750,000 patients per year, and a social cost estimated at $50 billion. Current therapeutics are targeted at restoring blood flow rather than on preventing the actual mechanisms associated with neuronal cell death. Here, we show that, following transient (2 h) middle cerebral artery occlusion (tMCAO) in male, Wistar rats, neuronal damage determined using MAP-2 staining increased progressively after the tMCAO. Notably, such neuronal degeneration was first associated with a decrease in p-Akt in both the focus and penumbra of the infarct region and, later with an increase in cytosolic cytochrome C levels in cortical neurons in the infarct area. These findings implicate that Akt alterations and consequent release of cytochrome C are involved in neuronal death. To further address this issue, NXY-059 (disodium 4-[(tert.-butylimino)methyl]benzene-1,3-disulfonate N-oxide) administered i.v. (30 mg/kg bolus, followed by 30 mg/kg/h infusion for up to 24 h), commencing 1 h after reperfusion, not only prevented the increase in infarct area but also attenuated the postreperfusion increase in neuronal cytosolic cytochrome C and the postperfusion decrease in neuronal p-Akt. Thus, NXY-059, by preventing mitochondrial cytochrome C release by maintaining activation of the Akt pathway, appears to protect neurons from damage after ischemia.