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1.  DNA methylation, microRNAs, and their crosstalk as potential biomarkers in hepatocellular carcinoma 
Epigenetic alterations have been identified as a major characteristic in human cancers. Advances in the field of epigenetics have contributed significantly in refining our knowledge of molecular mechanisms underlying malignant transformation. DNA methylation and microRNA expression are epigenetic mechanisms that are widely altered in human cancers including hepatocellular carcinoma (HCC), the third leading cause of cancer related mortality worldwide. Both DNA methylation and microRNA expression patterns are regulated in developmental stage specific-, cell type specific- and tissue-specific manner. The aberrations are inferred in the maintenance of cancer stem cells and in clonal cell evolution during carcinogenesis. The availability of genome-wide technologies for DNA methylation and microRNA profiling has revolutionized the field of epigenetics and led to the discovery of a number of epigenetically silenced microRNAs in cancerous cells and primary tissues. Dysregulation of these microRNAs affects several key signalling pathways in hepatocarcinogenesis suggesting that modulation of DNA methylation and/or microRNA expression can serve as new therapeutic targets for HCC. Accumulative evidence shows that aberrant DNA methylation of certain microRNA genes is an event specifically found in HCC which correlates with unfavorable outcomes. Therefore, it can potentially serve as a biomarker for detection as well as for prognosis, monitoring and predicting therapeutic responses in HCC.
doi:10.3748/wjg.v20.i24.7894
PMCID: PMC4069317  PMID: 24976726
DNA methylation; MicroRNA; Epigenetics; Hepatocellular carcinoma; Biomarker
2.  Is Upregulation of BCL2 a Determinant of Tumor Development Driven by Inactivation of CDH1/E-Cadherin? 
PLoS ONE  2013;8(8):e73062.
Inactivation of CDH1, encoding E-cadherin, promotes cancer initiation and progression. According to a newly proposed molecular mechanism, loss of E-cadherin triggers an upregulation of the anti-apoptotic oncoprotein BCL2. Conversely, reconstitution of E-cadherin counteracts overexpression of BCL2. This reciprocal regulation is thought to be critical for early tumor development. We determined the relevance of this new concept in human infiltrating lobular breast cancer (ILBC), the prime tumor entity associated with CDH1 inactivation. BCL2 expression was examined in human ILBC cell lines (IPH-926, MDA-MB-134, SUM-44) harboring deleterious CDH1 mutations. To test for an intact regulatory axis between E-cadherin and BCL2, wild-type E-cadherin was reconstituted in ILBC cells by ectopic expression. Moreover, BCL2 and E-cadherin were evaluated in primary invasive breast cancers and in synchronous lobular carcinomas in situ (LCIS). MDA-MB-134 and IPH-926 showed little or no BCL2 expression, while SUM-44 ILBC cells were BCL2-positive. Reconstitution of E-cadherin failed to impact on BCL2 expression in all cell lines tested. Primary ILBCs were almost uniformly E-cadherin-negative (97%) and were frequently BCL2-negative (46%). When compared with an appropriate control group, ILBCs showed a trend towards an increased frequency of BCL2-negative cases (P = 0.064). In terminal duct-lobular units affected by LCIS, the E-cadherin-negative neoplastic component showed a similar or a reduced BCL2-immunoreactivity, when compared with the adjacent epithelium. In conclusion, upregulation of BCL2 is not involved in lobular breast carcinogenesis and is unlikely to represent an important determinant of tumor development driven by CDH1 inactivation.
doi:10.1371/journal.pone.0073062
PMCID: PMC3758309  PMID: 24023670
3.  Loss of Imprinting and Allelic Switching at the DLK1-MEG3 Locus in Human Hepatocellular Carcinoma 
PLoS ONE  2012;7(11):e49462.
Deregulation of imprinted genes is an important molecular mechanism contributing to the development of cancer in humans. However, knowledge about imprinting defects in human hepatocellular carcinoma (HCC), the third leading cause of cancer mortality worldwide, is still limited. Therefore, a systematic meta-analysis of the expression of 223 imprinted loci in human HCC was initiated. This screen revealed that the DLK1-MEG3 locus is frequently deregulated in HCC. Deregulation of DLK1 and MEG3 expression accompanied by extensive aberrations in DNA methylation could be confirmed experimentally in an independent series of human HCC (n = 40) in more than 80% of cases. Loss of methylation at the DLK1-MEG3 locus correlates linearly with global loss of DNA methylation in HCC (r2 = 0.63, p<0.0001). Inhibition of DNMT1 in HCC cells using siRNA led to a reduction in MEG3-DMR methylation and concomitant increase in MEG3 RNA expression. Allele-specific expression analysis identified loss of imprinting in 10 out of 31 informative samples (32%), rendering it one of the most frequent molecular defects in human HCC. In 2 cases unequivocal gain of bi-allelic expression accompanied by substantial loss of methylation at the IG-DMR could be demonstrated. In 8 cases the tumour cells displayed allelic switching by mono-allelic expression of the normally imprinted allele. Allelic switching was accompanied by gains or losses of DNA methylation primarily at IG-DMR1. Analysis of 10 hepatocellular adenomas (HCA) and 5 cases of focal nodular hyperplasia (FNH) confirmed that this epigenetic instability is specifically associated with the process of malignant transformation and not linked to increased proliferation per se. This widespread imprint instability in human HCC has to be considered in order to minimize unwanted side-effects of therapeutic approaches targeting the DNA methylation machinery. It might also serve in the future as predictive biomarker and for monitoring response to epigenetic therapy.
doi:10.1371/journal.pone.0049462
PMCID: PMC3493531  PMID: 23145177
4.  Limited Value of KAI1/CD82 Protein Expression as a Prognostic Marker in Human Gastric Cancer 
Disease markers  2012;32(6):337-342.
The cell surface glycoprotein KAI1/CD82 suppresses tumor growth and metastasis in animal models. This study aimed to evaluate the prognostic relevance of KAI1/CD82 protein expression in human gastric cancer. Primary gastric carcinomas (n = 271) with amean clinical follow-up time of 48months were immunostained using the monoclonal anti-KAI1/CD82 antibody G2. Staining was evaluated as negative versus positive for statistical analysis. KAI1/CD82 immunoreactivity was absent in 103/271 (38%) cases. There was a trend towards KAI1/CD82 negativity in poorly differentiated cases (p = 0.0679). Moreover, KAI1/CD82-negative carcinomas were associated with a higher pT status (p = 0.0222), metastatic lymph node involvement (p = 0.0018) and a higher clinical tumor stage (p = 0.0050). The median overall survival times of KAI1/CD82-negative and KAI1/CD82-positive gastric carcinomas were 20 and 37 months, respectively (p = 0.2305). These results are in line with the proposed function of KAI1/CD82 as a suppressor of tumor growth and metastasis. However, these data suggest that KAI1/CD82, as detected by immunohistochemistry, is of limited value as a prognostic marker for gastric cancer in routine histological workup.
doi:10.3233/DMA-2012-0896
PMCID: PMC3826928  PMID: 22684230
Gastric cancer; prognostic markers; metastasis; immunohistochemistry
5.  Nuclear Kaiso Expression Is Associated with High Grade and Triple-Negative Invasive Breast Cancer 
PLoS ONE  2012;7(5):e37864.
Kaiso is a BTB/POZ transcription factor that is ubiquitously expressed in multiple cell types and functions as a transcriptional repressor and activator. Little is known about Kaiso expression and localization in breast cancer. Here, we have related pathological features and molecular subtypes to Kaiso expression in 477 cases of human invasive breast cancer. Nuclear Kaiso was predominantly found in invasive ductal carcinoma (IDC) (p = 0.007), while cytoplasmic Kaiso expression was linked to invasive lobular carcinoma (ILC) (p = 0.006). Although cytoplasmic Kaiso did not correlate to clinicopathological features, we found a significant correlation between nuclear Kaiso, high histological grade (p = 0.023), ERα negativity (p = 0.001), and the HER2-driven and basal/triple-negative breast cancers (p = 0.018). Interestingly, nuclear Kaiso was also abundant in BRCA1-associated breast cancer (p<0.001) and invasive breast cancer overexpressing EGFR (p = 0.019). We observed a correlation between nuclear Kaiso and membrane-localized E-cadherin and p120-catenin (p120) (p<0.01). In contrast, cytoplasmic p120 strongly correlated with loss of E-cadherin and low nuclear Kaiso (p = 0.005). We could confirm these findings in human ILC cells and cell lines derived from conditional mouse models of ILC. Moreover, we present functional data that substantiate a mechanism whereby E-cadherin controls p120-mediated relief of Kaiso-dependent gene repression. In conclusion, our data indicate that nuclear Kaiso is common in clinically aggressive ductal breast cancer, while cytoplasmic Kaiso and a p120-mediated relief of Kaiso-dependent transcriptional repression characterize ILC.
doi:10.1371/journal.pone.0037864
PMCID: PMC3360634  PMID: 22662240
6.  Quantitative cross-validation and content analysis of the 450k DNA methylation array from Illumina, Inc. 
BMC Research Notes  2012;5:210.
Background
The newly released 450k DNA methylation array from Illumina, Inc. offers the possibility to analyze more than 480,000 individual CpG sites in a user friendly standardized format. In this study the relationship between the β-values provided by the Illumina, Inc. array for each individual CpG dinucleotide and the quantitative methylation levels obtained by pyrosequencing were analyzed. In addition, the representation of microRNA genes and imprinted loci on the Illumina, Inc. array was assessed in detail. Genomic DNA from 4 human breast cancer cell lines (IPH-926, HCC1937, MDA-MB-134, PMC42) and 18 human breast cancer specimens as well as 4 normal mammary epithelial fractions was analyzed on 450k DNA methylation arrays. The β-values for 692 individual CpG sites from 62 different genes were cross-validated using conventional quantitative pyrosequencing.
Findings
The newly released 450k methylation array from Illumina, Inc. shows a high concordance with quantitative pyrosequencing if identical CpG sites are analyzed in cell lines (Spearman r = 0.88, p ≪ 0.0001), which is somewhat reduced in primary tumor specimens (Spearman r = 0.86, p ≪ 0.0001). 80.7% of the CpG sites show an absolute difference in methylation level of less than 15 percentage points. If different CpG sites in the same CpG islands are targeted the concordance is lower (r = 0.83 in cell lines and r = 0.7 in primary tumors). The number of CpG sites representing microRNA genes and imprinted loci is very heterogeneous (range: 1 – 70 CpG sites for microRNAs and 1 – 288 for imprinted loci).
Conclusions
The newly released 450k methylation array from Illumina, Inc. provides a genome-wide quantitative representation of DNA methylation aberrations in a convenient format. Overall, the congruence with pyrosequencing data is very good. However, for individual loci one should be careful to translate the β-values directly into percent methylation levels.
doi:10.1186/1756-0500-5-210
PMCID: PMC3420245  PMID: 22546179
7.  Decentral gene expression analysis for ER+/Her2− breast cancer: results of a proficiency testing program for the EndoPredict assay 
Virchows Archiv  2012;460(3):251-259.
Gene expression profiles provide important information about the biology of breast tumors and can be used to develop prognostic tests. However, the implementation of quantitative RNA-based testing in routine molecular pathology has not been accomplished, so far. The EndoPredict assay has recently been described as a quantitative RT-PCR-based multigene expression test to identify a subgroup of hormone–receptor-positive tumors that have an excellent prognosis with endocrine therapy only. To transfer this test from bench to bedside, it is essential to evaluate the test–performance in a multicenter setting in different molecular pathology laboratories. In this study, we have evaluated the EndoPredict (EP) assay in seven different molecular pathology laboratories in Germany, Austria, and Switzerland. A set of ten formalin-fixed paraffin-embedded tumors was tested in the different labs, and the variance and accuracy of the EndoPredict assays were determined using predefined reference values. Extraction of a sufficient amount of RNA and generation of a valid EP score was possible for all 70 study samples (100%). The EP scores measured by the individual participants showed an excellent correlation with the reference values, respectively, as reflected by Pearson correlation coefficients ranging from 0.987 to 0.999. The Pearson correlation coefficient of all values compared to the reference value was 0.994. All laboratories determined EP scores for all samples differing not more than 1.0 score units from the pre-defined references. All samples were assigned to the correct EP risk group, resulting in a sensitivity and specificity of 100%, a concordance of 100%, and a kappa of 1.0. Taken together, the EndoPredict test could be successfully implemented in all seven participating laboratories and is feasible for reliable decentralized assessment of gene expression in luminal breast cancer.
doi:10.1007/s00428-012-1204-4
PMCID: PMC3306560  PMID: 22371223
Breast cancer; Prognosis; mRNA; Quality control
8.  Acute-Phase Protein α1-Antitrypsin Inhibits Neutrophil Calpain I and Induces Random Migration 
Molecular Medicine  2011;17(9-10):865-874.
A rapid recruitment of neutrophils to sites of injury or infection is a hallmark of the inflammatory response and is required for effective host defense against pathogenic stimuli. However, neutrophil-mediated inflammation can also lead to chronic tissue destruction; therefore, a better understanding of the mechanisms underlying neutrophil influx and activation is of critical importance. We have previously shown that the acute phase protein α1-antitrypsin (AAT) inhibits neutrophil chemotaxis. In this study, we examine mechanisms related to the effect of AAT on neutrophil responses. We report a previously unknown function of AAT to inactivate calpain I (μ-calpain) and to induce a rapid cell polarization and random migration. These effects of AAT coincided with a transient rise in intracellular calcium, increase in intracellular lipids, activation of the Rho GTPases, Rac1 and Cdc42, and extra-cellular signal-regulated kinase (ERK1/2). Furthermore, AAT caused a significant inhibition of nonstimulated as well as formyl-met-leu-phe (fMLP)-stimulated neutrophil adhesion to fibronectin, strongly inhibited lipopolysaccharide-induced IL-8 release and slightly delayed neutrophil apoptosis. The results presented here broaden our understanding of the regulation of calpain-related neutrophil functional activities, and provide the impetus for new studies to define the role of AAT and other acute phase proteins in health and disease.
doi:10.2119/molmed.2011.00089
PMCID: PMC3188872  PMID: 21494752
9.  GDF-15 is abundantly expressed in plexiform lesions in patients with pulmonary arterial hypertension and affects proliferation and apoptosis of pulmonary endothelial cells 
Respiratory Research  2011;12(1):62.
Background
Growth-differentiation factor-15 (GDF-15) is a stress-responsive, transforming growth factor-β-related cytokine, which has recently been reported to be elevated in serum of patients with idiopathic pulmonary arterial hypertension (IPAH). The aim of the study was to examine the expression and biological roles of GDF-15 in the lung of patients with pulmonary arterial hypertension (PAH).
Methods
GDF-15 expression in normal lungs and lung specimens of PAH patients were studied by real-time RT-PCR and immunohistochemistry. Using laser-assisted micro-dissection, GDF-15 expression was further analyzed within vascular compartments of PAH lungs. To elucidate the role of GDF-15 on endothelial cells, human pulmonary microvascular endothelial cells (HPMEC) were exposed to hypoxia and laminar shear stress. The effects of GDF-15 on the proliferation and cell death of HPMEC were studied using recombinant GDF-15 protein.
Results
GDF-15 expression was found to be increased in lung specimens from PAH patients, com-pared to normal lungs. GDF-15 was abundantly expressed in pulmonary vascular endothelial cells with a strong signal in the core of plexiform lesions. HPMEC responded with marked upregulation of GDF-15 to hypoxia and laminar shear stress. Apoptotic cell death of HPMEC was diminished, whereas HPMEC proliferation was either increased or decreased depending of the concentration of recombinant GDF-15 protein.
Conclusions
GDF-15 expression is increased in PAH lungs and appears predominantly located in vascular endothelial cells. The expression pattern as well as the observed effects on proliferation and apoptosis of pulmonary endothelial cells suggest a role of GDF-15 in the homeostasis of endothelial cells in PAH patients.
doi:10.1186/1465-9921-12-62
PMCID: PMC3113721  PMID: 21548946
10.  Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution 
BMC Biotechnology  2011;11:6.
Background
New high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparison with existing validated DNA methylation analysis methods is not yet available. Therefore, a systematic cross-validation of 454 sequencing and conventional pyrosequencing, both of which offer exact quantification of methylation levels with a single CpG dinucleotide resolution, was performed.
Results
To this end the methylation patterns of 12 loci (GSTπ1, p16INK4a, RASSF1A, SOCS1, MAL, hsa-mir-1-1, hsa-mir-9-3, hsa-mir-34a, hsa-mir-596, hsa-mir-663, MINT31, and LINE-1) were analyzed in ten primary hepatocellular carcinoma specimens. After applying stringent quality control criteria, 35749 sequences entered further analysis. The methylation level of individual CpG dinucleotides obtained by 454 sequencing was systematically compared with the corresponding values obtained by conventional pyrosequencing. Statistical analyses revealed an excellent concordance of methylation levels for all individual CpG dinucleotides under study (r2 = 0.927).
Conclusions
Our results confirm that 454 sequencing of bisulfite treated genomic DNA provides reliable high quality quantitative methylation data and identify MAL, hsa-mir-9-3, hsa-mir-596, and hsa-mir-663 as new targets of aberrant DNA methylation in human hepatocelluar carcinoma. In addition, the single molecule resolution of 454 sequencing provides unprecedented information about the details of DNA methylation pattern heterogeneity in clinical samples.
doi:10.1186/1472-6750-11-6
PMCID: PMC3032674  PMID: 21235780
11.  Frequent and Distinct Aberrations of DNA Methylation Patterns in Fibrolamellar Carcinoma of the Liver 
PLoS ONE  2010;5(10):e13688.
Background
Gene silencing due to aberrant DNA methylation is a frequent event in hepatocellular carcinoma (HCC) and also in hepatocellular adenoma (HCA). However, very little is known about epigenetic defects in fibrolamellar carcinoma (FLC), a rare variant of hepatocellular carcinoma that displays distinct clinical and morphological features.
Methodology/Principal Findings
We analyzed the methylation status of the APC, CDH1, cyclinD2, GSTπ1, hsa-mir-9-1, hsa-mir-9-2, and RASSF1A gene in a series of 15 FLC and paired normal liver tissue specimens by quantitative high-resolution pyrosequencing. Results were compared with common HCC arising in non-cirrhotic liver (n = 10). Frequent aberrant hypermethylation was found for the cyclinD2 (19%) and the RASSF1A (38%) gene as well as for the microRNA genes mir-9-1 (13%) and mir-9-2 (33%). In contrast to common HCC the APC and CDH1 (E-cadherin) genes were found devoid of any DNA methylation in FLC, whereas the GSTπ1 gene showed comparable DNA methylation in tumor and surrounding tissue at a moderate level. Changes in global DNA methylation level were measured by analyzing methylation status of the highly repetitive LINE-1 sequences. No evidence of global hypomethylation could be found in FLCs, whereas HCCs without cirrhosis showed a significant reduction in global methylation level as described previously.
Conclusions
FLCs display frequent and distinct gene-specific hypermethylation in the absence of significant global hypomethylation indicating that these two epigenetic aberrations are induced by different pathways and that full-blown malignancy can develop in the absence of global loss of DNA methylation. Only quantitative DNA methylation detection methodology was able to identify these differences.
doi:10.1371/journal.pone.0013688
PMCID: PMC2966398  PMID: 21060828
12.  Identification of differentially expressed microRNAs in human male breast cancer 
BMC Cancer  2010;10:109.
Background
The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases.
Methods
The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.
Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer.
Results
Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer.
Conclusions
Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.
doi:10.1186/1471-2407-10-109
PMCID: PMC2850898  PMID: 20331864
13.  Differential roles of NHERF1, NHERF2, and PDZK1 in regulating CFTR-mediated intestinal anion secretion in mice 
The epithelial anion channel CFTR interacts with multiple PDZ domain–containing proteins. Heterologous expression studies have demonstrated that the Na+/H+ exchanger regulatory factors, NHERF1, NHERF2, and PDZK1 (NHERF3), modulate CFTR membrane retention, conductivity, and interactions with other transporters. To study their biological roles in vivo, we investigated CFTR-dependent duodenal HCO3– secretion in mouse models of Nherf1, Nherf2, and Pdzk1 loss of function. We found that Nherf1 ablation strongly reduced basal as well as forskolin-stimulated (FSK-stimulated) HCO3– secretory rates and blocked β2-adrenergic receptor (β2-AR) stimulation. Conversely, Nherf2–/– mice displayed augmented FSK-stimulated HCO3– secretion. Furthermore, although lysophosphatidic acid (LPA) inhibited FSK-stimulated HCO3– secretion in WT mice, this effect was lost in Nherf2–/– mice. Pdzk1 ablation reduced basal, but not FSK-stimulated, HCO3– secretion. In addition, laser microdissection and quantitative PCR revealed that the β2-AR and the type 2 LPA receptor were expressed together with CFTR in duodenal crypts and that colocalization of the β2-AR and CFTR was reduced in the Nherf1–/– mice. These data suggest that the NHERF proteins differentially modulate duodenal HCO3– secretion: while NHERF1 is an obligatory linker for β2-AR stimulation of CFTR, NHERF2 confers inhibitory signals by coupling the LPA receptor to CFTR.
doi:10.1172/JCI35541
PMCID: PMC2648694  PMID: 19221439
14.  Reliable microRNA profiling in routinely processed formalin-fixed paraffin-embedded breast cancer specimens using fluorescence labelled bead technology 
BMC Biotechnology  2008;8:90.
Background
During the last years the analysis of microRNA expression patterns has led to completely new insights into cancer biology. Furthermore, these patterns are a very promising tool for the development of new diagnostic and prognostic markers. However, most human tumour samples for which long term clinical records are available exist only as formalin-fixed paraffin-embedded specimens. Therefore, the aim of this study was to examine the feasibility of microRNA profiling studies in routinely processed formalin-fixed paraffin-embedded human breast cancer specimens using fluorescence labelled bead technology.
Results
A statistically highly significant correlation (Spearman r: 0.78 – 0.90, p < 0.0001) was observed for the expression of 319 microRNAs in routinely processed FFPE breast cancer specimens and paired fresh frozen tissue samples (n = 5). Results were confirmed in a larger series analyzing a selection of 10 microRNAs reported to be deregulated in breast cancer (n = 12). The expression pattern of 3 microRNAs was independently validated in this cohort using real-time RT-PCR technology.
Conclusion
Comprehensive microRNA expression patterns can be reliably derived from routinely processed FFPE breast cancer specimens using fluorescence labelled bead technology.
doi:10.1186/1472-6750-8-90
PMCID: PMC2605753  PMID: 19038028

Results 1-14 (14)