Bálint Péterfia

Ablation of the decorin gene enhances experimental hepatic fibrosis and impairs hepatic healing in mice

Accumulation of connective tissue is a typical feature of chronic liver diseases. Decorin, a small leucine-rich proteoglycan, regulates collagen fibrillogenesis during development, and by directly blocking the bioactivity of transforming growth factor-β1 (TGFβ1), it exerts a protective effect against fibrosis. However, no in vivo investigations on the role of decorin in liver have been performed before. In this study we used decorin-null (Dcn−/−) mice to establish the role of decorin in experimental liver fibrosis and repair. Not only the extent of experimentally induced liver fibrosis was more severe in Dcn−/− animals, but also the healing process was significantly delayed vis-à-vis wild-type mice. Collagen I, III, and IV mRNA levels in Dcn−/− livers were higher than those of wild-type livers only in the first 2 months, but no difference was observed after 4 months of fibrosis induction, suggesting that the elevation of these proteins reflects a specific impairment of their degradation. Gelatinase assays confirmed this hypothesis as we found decreased MMP-2 and MMP-9 activity and higher expression of TIMP-1 and PAI-1 mRNA in Dcn−/− livers. In contrast, at the end of the recovery phase increased production rather than impaired degradation was found to be responsible for the excessive connective tissue deposition in livers of Dcn−/− mice. Higher expression of TGFβ1-inducible early responsive gene in decorin-null livers indicated enhanced bioactivity of TGFβ1 known to upregulate TIMP-1 and PAI-1 as well. Moreover, two main axes of TGFβ1-evoked signaling pathways were affected by decorin deficiency, namely the Erk1/2 and Smad3 were activated in Dcn−/− samples, whereas no significant difference in phospho-Smad2 was observed between mice with different genotypes. Collectively, our results indicate that the lack of decorin favors the development of hepatic fibrosis and attenuates its subsequent healing process at least in part by affecting the bioactivity of TGFβ1.

Syndecan-1 Enhances Proliferation, Migration and Metastasis of HT-1080 Cells in Cooperation with Syndecan-2

Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression.

Genome-wide screening of genes regulated by DNA methylation in colon cancer development.

Tumorigenesis is accompanied by changes in the DNA methylation pattern. Our aim was to test a novel approach for identification of transcripts at whole transcript level which are regulated by DNA methylation. Our approach is based on comparison of data obtained from transcriptome profiling of primary human samples and in vitro cell culture models. Epithelial cells were collected by LCM from normal, adenoma, and tumorous colonic samples. Using gene expression analysis, we identified downregulated genes in the tumors compared to normal tissues. In parallel 3000 upregulated genes were determined in HT-29 colon adenocarcinoma cell culture model after DNA demethylation treatment. Of the 2533 transcripts showing reduced expression in the tumorous samples, 154 had increased expression as a result of DNA demethylation treatment. Approximately 2/3 of these genes had decreased expression already in the adenoma samples. Expression of five genes (GCG, NMES-1, LRMP, FAM161B and PTGDR), was validated using RT-PCR. PTGDR showed ambiguous results, therefore it was further studied to verify the extent of DNA methylation and its effect on the protein level. Results confirmed that our approach is suitable for genome-wide screening of genes which are regulated or inactivated by DNA methylation. Activity of these genes possibly interferes with tumor progression, therefore genes identified can be key factors in the formation and in the progression of the disease.

Effect of syndecan-1 overexpression on mesenchymal tumour cell proliferation with focus on different functional domains

Objectives:  Syndecan‐1 is a transmembrane proteoglycan involved in various biological processes. Its extracellular, transmembrane and cytoplasmic domains may all participate in signal transduction. The aim of this study was to investigate the biological roles of these domains of syndecan‐1.

Comprehensive DNA Methylation Analysis Reveals a Common Ten-Gene Methylation Signature in Colorectal Adenomas and Carcinomas

Microarray analysis of promoter hypermethylation provides insight into the role and extent of DNA methylation in the development of colorectal cancer (CRC) and may be co-monitored with the appearance of driver mutations. Colonic biopsy samples were obtained endoscopically from 10 normal, 23 adenoma (17 low-grade (LGD) and 6 high-grade dysplasia (HGD)), and 8 ulcerative colitis (UC) patients (4 active and 4 inactive). CRC samples were obtained from 24 patients (17 primary, 7 metastatic (MCRC)), 7 of them with synchronous LGD. Field effects were analyzed in tissues 1 cm (n = 5) and 10 cm (n = 5) from the margin of CRC. Tissue materials were studied for DNA methylation status using a 96 gene panel and for KRAS and BRAF mutations. Expression levels were assayed using whole genomic mRNA arrays. SFRP1 was further examined by immunohistochemistry. HT29 cells were treated with 5-aza-2’ deoxycytidine to analyze the reversal possibility of DNA methylation. More than 85% of tumor samples showed hypermethylation in 10 genes (SFRP1, SST, BNC1, MAL, SLIT2, SFRP2, SLIT3, ALDH1A3, TMEFF2, WIF1), whereas the frequency of examined mutations were below 25%. These genes distinguished precancerous and cancerous lesions from inflamed and healthy tissue. The mRNA alterations that might be caused by systematic methylation could be partly reversed by demethylation treatment. Systematic changes in methylation patterns were observed early in CRC carcinogenesis, occuring in precursor lesions and CRC. Thus we conclude that DNA hypermethylation is an early and systematic event in colorectal carcinogenesis, and it could be potentially reversed by systematic demethylation therapy, but it would need more in vitro and in vivo experiments to support this theory.

SMARCB1 protein and mRNA loss is not caused by promoter and histone hypermethylation in epithelioid sarcoma

About 10% of epithelioid sarcomas have biallelic mutation of the SMARCB1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1) gene resulting in a lack of this nuclear protein. It has been suggested that SMARCB1 may be silenced by epigenetic changes in the remaining 90% of tumors. Thus, we hypothesized that the promoter of SMARCB1 is hypermethylated. We also examined SMARCB1 mRNA level to determine if a post-translational change was possible. Thirty-six cases of epithelioid sarcomas were studied. Immunohistochemistry and mutation analysis of the SMARCB1 gene were performed to select appropriate cases. Methylation status was assessed by methylation-specific PCR. Laser capture microdissection of tumor cells followed by real-time PCR was applied to examine the expression of SMARCB1 mRNA. Of 36 epithelioid sarcomas, 31 (86%) displayed a lack of SMARCB1 nuclear protein. In all, 4 (13%) of 31 SMARCB1-negative cases harbored biallelic deletion while 9 (33%) cases showed single-allelic deletion. One (4%) frameshift deletion of exon 3 and one point mutation of exon 7 were also found. In 16 (59%) cases, both alleles were intact. Altogether, 25/31 (81%) SMARCB1-negative cases had at least one intact allele. None of these cases demonstrated promoter hypermethylation. Low levels of SMARCB1 mRNA were found in all cases with tumor tissue extracted RNA (because of the minimal normal cell contamination) but no mRNA could be detected in laser dissected cases (containing only tumor cells). Enhancer of zeste homolog 2 (EZH2) overexpression was not characteristic of epithelioid sarcoma. Thus, loss of SMARCB1 expression in epithelioid sarcoma is caused neither by DNA hypermethylation nor by post-translational modifications. Most likely it is the microRNA destruction of SMARCB1 mRNA but further investigations are needed to elucidate this issue.

Myofibroblast-derived SFRP1 as potential inhibitor of colorectal carcinoma field effect.

Epigenetic changes of stromal-epithelial interactions are of key importance in the regulation of colorectal carcinoma (CRC) cells and morphologically normal, but genetically and epigenetically altered epithelium in normal adjacent tumor (NAT) areas. Here we demonstrated retained protein expression of well-known Wnt inhibitor, secreted frizzled-related protein 1 (SFRP1) in stromal myofibroblasts and decreasing epithelial expression from NAT tissues towards the tumor. SFRP1 was unmethylated in laser microdissected myofibroblasts and partially hypermethylated in epithelial cells in these areas. In contrast, we found epigenetically silenced myofibroblast-derived SFRP1 in CRC stroma. Our results suggest that the myofibroblast-derived SFRP1 protein might be a paracrine inhibitor of epithelial proliferation in NAT areas and loss of this signal may support tumor proliferation in CRC.

DNA hypermethylation and decreased mRNA expression of MAL, PRIMA1, PTGDR and SFRP1 in colorectal adenoma and cancer

BackgroundColorectal cancer (CRC) development is accompanied by changes in expression for several genes; but the details of the underlying regulatory procesess remain unknown. Our aims were to assess the role of epigenetic processes in tumour formation and to identify characteristic DNA methylation and miRNA alterations in the colorectal adenoma-carcinoma sequence.MethodsWhole genome expression profiling was performed on colonic biopsy samples (49 healthy normal, 49 colorectal adenoma (AD), 49 CRC); on laser capture microdissected (LCM) epithelial and stromal cells from 6 CRC-normal adjacent tissue (NAT) samples pairs, and on demethylated human CRC cell lines using HGU133 Plus 2.0 microarrays (Affymetrix). Methylation status of genes with gradually altering expression along the AD-CRC sequence was further analysed on 10–10 macrodissected and 5–5 LCM samples from healthy colon, from adenoma and from CRC biopsy samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes was performed with miRWALK algorithm, miRNA expression was analysed on 3 CRC-NAT sample pairs and 3 adenoma tissue samples using the Human Panel I + II (Exiqon). SFRP1 immunohistochemistry experiments were performed.ResultsA set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p < 0.01) in the biopsy samples along the adenoma-carcinoma sequence. Three of those (COL1A2, SFRP2, SOCS3) showed hypermethylation and THBS2 showed hypomethylation both in AD and in CRC samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p < 0.01) upregulated in adenoma and tumour samples compared to the healthy colonic tissue controls and could explain the altered expression of genes for which DNA methylation changes do not appear to play role (e.g. BCL2, MAL, PTGS2). Demethylation treatment could upregulate gene expression of genes that were found to be hypermethylated in human CRC tissue samples. Decreasing protein levels of SFRP1 was also observed along the adenoma-carcinoma sequence.ConclusionHypermethylation of the selected markers (MAL, PRIMA1, PTGDR and SFRP1) can result in reduced gene expression and may contribute to the formation of colorectal cancer.

Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer.

ABSTRACT The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2′-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.

Aging related methylation influences the gene expression of key control genes in colorectal cancer and adenoma

AIM To analyze colorectal carcinogenesis and age-related DNA methylation alterations of gene sequences associated with epigenetic clock CpG sites. METHODS In silico DNA methylation analysis of 353 epigenetic clock CpG sites published by Steve Horvath was performed using methylation array data for a set of 123 colonic tissue samples [64 colorectal cancer (CRC), 42 adenoma, 17 normal; GEO accession number: GSE48684]. Among the differentially methylated age-related genes, secreted frizzled related protein 1 (SFRP1) promoter methylation was further investigated in colonic tissue from 8 healthy adults, 19 normal children, 20 adenoma and 8 CRC patients using bisulfite-specific PCR followed by methylation-specific high resolution melting (MS-HRM) analysis. mRNA expression of age-related “epigenetic clock” genes was studied using Affymetrix HGU133 Plus2.0 whole transcriptome data of 153 colonic biopsy samples (49 healthy adult, 49 adenoma, 49 CRC, 6 healthy children) (GEO accession numbers: GSE37364, GSE10714, GSE4183, GSE37267). Whole promoter methylation analysis of genes showing inverse DNA methylation-gene expression data was performed on 30 colonic samples using methyl capture sequencing. RESULTS Fifty-seven age-related CpG sites including hypermethylated PPP1R16B, SFRP1, SYNE1 and hypomethylated MGP, PIPOX were differentially methylated between CRC and normal tissues (P < 0.05, Δβ ≥ 10%). In the adenoma vs normal comparison, 70 CpG sites differed significantly, including hypermethylated DKK3, SDC2, SFRP1, SYNE1 and hypomethylated CEMIP, SPATA18 (P < 0.05, Δβ ≥ 10%). In MS-HRM analysis, the SFRP1 promoter region was significantly hypermethylated in CRC (55.0% ± 8.4 %) and adenoma tissue samples (49.9% ± 18.1%) compared to normal adult (5.2% ± 2.7%) and young (2.2% ± 0.7%) colonic tissue (P < 0.0001). DNA methylation of SFRP1 promoter was slightly, but significantly increased in healthy adults compared to normal young samples (P < 0.02). This correlated with significantly increased SFRP1 mRNA levels in children compared to normal adult samples (P < 0.05). In CRC tissue the mRNA expression of 117 age-related genes were changed, while in adenoma samples 102 genes showed differential expression compared with normal colonic tissue (P < 0.05, logFC > 0.5). The change of expression for several genes including SYNE1, CLEC3B, LTBP3 and SFRP1, followed the same pattern in aging and carcinogenesis, though not for all genes (e.g., MGP). CONCLUSION Several age-related DNA methylation alterations can be observed during CRC development and progression affecting the mRNA expression of certain CRC- and adenoma-related key control genes.