Péter Hollósi

Syndecan-1 and FGF-2, but not FGF receptor-1, share a common transport route and co-localize with heparanase in the nuclei of mesenchymal tumor cells

Syndecan-1 forms complexes with growth factors and their cognate receptors in the cell membrane. We have previously reported a tubulin-mediated translocation of syndecan-1 to the nucleus. The transport route and functional significance of nuclear syndecan-1 is still incompletely understood. Here we investigate the sub-cellular distribution of syndecan-1, FGF-2, FGFR-1 and heparanase in malignant mesenchymal tumor cells, and explore the possibility of their coordinated translocation to the nucleus. To elucidate a structural requirement for this nuclear transport, we have transfected cells with a syndecan-1/EGFP construct or with a short truncated version containing only the tubulin binding RMKKK sequence. The sub-cellular distribution of the EGFP fusion proteins was monitored by fluorescence microscopy. Our data indicate that syndecan-1, FGF-2 and heparanase co-localize in the nucleus, whereas FGFR-1 is enriched mainly in the perinuclear area. Overexpression of syndecan-1 results in increased nuclear accumulation of FGF-2, demonstrating the functional importance of syndecan-1 for this nuclear transport. Interestingly, exogenously added FGF-2 does not follow the route taken by endogenous FGF-2. Furthermore, we prove that the RMKKK sequence of syndecan-1 is necessary and sufficient for nuclear translocation, acting as a nuclear localization signal, and the Arginine residue is vital for this localization. We conclude that syndecan-1 and FGF-2, but not FGFR-1 share a common transport route and co-localize with heparanase in the nucleus, and this transport is mediated by the RMKKK motif in syndecan-1. Our study opens a new perspective in the proteoglycan field and provides more evidence of nuclear interactions of syndecan-1.

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.

Remodeling of extracellular matrix by normal and tumor-associated fibroblasts promotes cervical cancer progression.

BackgroundComparison of tissue microarray results of 29 cervical cancer and 27 normal cervix tissue samples using immunohistochemistry revealed considerable reorganization of the fibrillar stroma of these tumors.Preliminary densitometry analysis of laminin-1, α-smooth muscle actin (SMA) and fibronectin immunostaining demonstrated 3.8-fold upregulation of laminin-1 and 5.2-fold increase of SMA in the interstitial stroma, indicating that these proteins and the activated fibroblasts play important role in the pathogenesis of cervical cancer. In the present work we investigated the role of normal and tumor-associated fibroblasts.MethodsIn vitro models were used to throw light on the multifactorial process of tumor-stroma interaction, by means of studying the cooperation between tumor cells and fibroblasts. Fibroblasts from normal cervix and cervical cancers were grown either separately or in co-culture with CSCC7 cervical cancer cell line. Changes manifest in secreted glycoproteins, integrins and matrix metallo-proteases (MMPs) were explored.ResultsWhile normal fibroblasts produced components of interstitial matrix and TGF-β1 that promoted cell proliferation, cancer-associated fibroblasts (CAFs) synthesized ample amounts of laminin-1. The following results support the significance of laminin-1 in the invasion of CSCC7 cells: 1.) Tumor-associated fibroblasts produced more laminin-1 and less components of fibrillar ECM than normal cells; 2.) The production of laminin chains was further increased when CSCC7 cells were grown in co-culture with fibroblasts; 3.) CSCC7 cells were capable of increasing their laminin production; 4.) Tumor cells predominantly expressed integrin α6β4 laminin receptors and migrated towards laminin. The integrin profile of both normal and tumor-associated fibroblasts was similar, expressing receptors for fibronectin, vitronectin and osteopontin. MMP-7 secreted by CSCC7 cells was upregulated by the presence of normal fibroblasts, whereas MMP-2 produced mainly by fibroblasts was activated in the presence of CSCC7 cells.ConclusionsOur results indicate that in addition to degradation of the basement membrane, invasion of cervical cancer is accomplished by the remodeling of the interstitial stroma, which process includes decrease and partial replacement of fibronectin and collagens by a laminin-rich matrix.

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.

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.

Mutations of KRAS, NRAS, BRAF, EGFR, and PIK3CA genes in urachal carcinoma: Occurence and prognostic significance

Purpose Targeted therapy represents an attractive alternative for rare tumors such as urachal carcinoma (UrC). The aim of this study was to assess the mutations of the most commonly affected 5 genes in the targetable EGFR-pathway in UrC and comapre their frequencies to those of found in urothelial and colorectal cancer. Materials and Methods Mutational hot-spots of selected genes were tested in 22 UrC samples by pyrosequencing. Mutational patterns were compared to those published for colorectal and urothelial cancers. Furthermore, we sought correlations between mutations and clinicopathological and follow-up data. Results We found 11 mutations in 10 of 22 (45%) patients. The most frequently mutated gene was KRAS (27%) followed by BRAF (18%) and NRAS (5%), while no mutations were detected in the EGFR and PIK3CA genes. No correlation was found between the mutation status and clinicopathological parameters (Sheldon/Mayo stage, tumor grade, metastases). Furthermore, none of the mutations correlated with progression-free or overall survival. Conclusions The mutation pattern of UrC is more similar to colorectal than to urothelial cancer. However, the mutation characteristics of UrC seems to be unique suggesting that clinical decision-making for UrC cannot be simply adopted from urothelial or colorectal carcinoma. The high occurence of EGFR-pathway mutations warrants the testing for KRAS and BRAF mutations when considering anti-EGFR therapy in UrC.

Construction of a multiplex mutation hot spot PCR panel: The first step towards colorectal cancer genotyping on the GS Junior platform

Background: To support cancer therapy, development of low cost library preparation techniques for targeted next generation sequencing (NGS) is needed. In this study we designed and tested a PCR-based library preparation panel with limited target area for sequencing the top 12 somatic mutation hot spots in colorectal cancer on the GS Junior instrument. Materials and Methods: A multiplex PCR panel was designed to amplify regions of mutation hot spots in 12 selected genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53). Amplicons were sequenced on a GS Junior instrument using ligated and barcoded adaptors. Eight samples were sequenced in a single run. Colonic DNA samples (8 normal mucosa; 33 adenomas; 17 adenocarcinomas) as well as HT-29 and Caco-2 cell lines with known mutation profiles were analyzed. Variants found by the panel on APC, BRAF, KRAS and NRAS genes were validated by conventional sequencing. Results: In total, 34 kinds of mutations were detected including two novel mutations (FBXW7 c.1740:C>G and SMAD4 c.413C>G) that have not been recorded in mutation databases, and one potential germline mutation (APC). The most frequently mutated genes were APC, TP53 and KRAS with 30%, 15% and 21% frequencies in adenomas and 29%, 53% and 29% frequencies in carcinomas, respectively. In cell lines, all the expected mutations were detected except for one located in a homopolymer region. According to re-sequencing results sensitivity and specificity was 100% and 92% respectively. Conclusions: Our NGS-based screening panel denotes a promising step towards low cost colorectal cancer genotyping on the GS Junior instrument. Despite the relatively low coverage, we discovered two novel mutations and obtained mutation frequencies comparable to literature data. Additionally, as an advantage, this panel requires less template DNA than sequence capture colon cancer panels currently available for the GS Junior instrument.

Elsődleges genetikai vizsgálat Prader–Willi-szindróma igazolására

Absztrakt: Bevezetes: A nemzetkozi szakirodalmi adatok alapjan az SNRPN genlocus promoter regiojanak DNS-metilacios vizsgalata jelenleg a legerzekenyebb es leghatekonyabb kezdeti lepes a Prader–Willi-szindroma-gyanus betegek genetikai vizsgalatakor. Celkitűzes: Celunk egy egyszerű, megbizhato, konnyen hozzaferhető, elsődlegesen diagnosztikus, DNS-metilacion alapulo eljaras kidolgozasa volt Prader–Willi-szindroma igazolasara. Modszer: Vizsgalatunk soran az altalunk modositott, koltseghatekony, metilacioszenzitiv, nagy felbontasu olvadaspont-elemzeses technikat hasonlitottuk ossze a leginkabb elterjedt, koltseges metilaciospecifikus multiplex ligatiofuggő probaamplifikacios technikaval. Klinikailag a Prader–Willi-szindroma tobb tunetet mutato 17 gyermek DNS-metilacios vizsgalatat vegeztuk el sajat tervezesű primerekkel: biszulfitszekvenalo polimeraz-lancreakcio, metilacioszenzitiv nagy felbontasu olvadaspont-elemzes es kontrollkent metilaciospecifikus multiplex ligatiofuggő probaamplifikacio tortent. Eredme...

Methylation Status of CYP27B1 and IGF2 Correlate to BMI SDS in Children with Obesity

Objective: Worldwide increasing childhood obesity is due to interactions between environmental and genetic factors, linked together by epigenetic mechanisms such as DNA methylation. Methods: 82 obese children (>95th BMI percentile , age: 3-18 years) were included. Anthropometric data, metabolic parameters, 25-OH vitamin D (25OHD), and pubertal status were recorded, 24-hour blood pressure monitoring was performed. BMI standard deviation score (SDS) was calculated. Using candidate gene approach, obesity- (insulin-like growth factor 2 (IGF2), proopiomelanocortin (POMC)) and vitamin D metabolism-related genes (1-alfa-hydroxylase (CYP27B1), VDR) regulated by DNA methylation were selected. After isolating DNA from peripheral blood, bisulfite conversion, bisulfite specific polymerase chain reaction (BS-PCR), and pyrosequencing were carried out. Results: No significant correlation between 25-OHD and metabolic parameters and DNA methylation status, but a tendency of positive correlation between VDR methylation status and 25-OHD (r = 0.2053,p = 0.066) were observed. Significant positive correlations between BMI SDS and CYP27B1 hypermethylation (r = 0.2371,p = 0.0342) and a significant negative correlation between IGF2 hypomethylation and BMI SDS (r = -0.305,p = 0.0059) were found. Conclusions Rate of obesity shows correlation with DNA methylation. Hypomethylation of IGF2 and hypermethylation of CYP27B1 genes might positively influence the rate of BMI observed in obese children.

Abstract 422: DNA hypermethylation or upregulated miRNA21 expression potentially leads to decreased mRNA expression of COL1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in left-sided colorectal adenoma and cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: It is well known that beside genetic factors such as mutations, epigenetic mechanisms can also contribute to colorectal cancer formation and progression. Although several genes influenced by DNA methylation and miRNA expression alteration have been identified, our knowledge of the background of gene expression alterations during CRC development remains incomplete. Aims: Our aims were to identify DNA methylation markers and miRNAs playing role in left-sided CRC development on the basis of gene expression alterations along the adenoma-carcinoma formation. Materials & methods: Whole genome expression profiling was performed by using HGU133 Plus 2.0 microarrays (Affymetrix) on healthy colonic (n=49), colorectal adenoma (n=49) and left-sided CRC (n=49) biopsy samples and also on laser microdissected (LCM) epithelial and stromal cells from healthy (n=6) and CRC (n=6) samples. Methylation status of genes with gradually decreasing or increasing expression along the adenoma-carcinoma sequence were analyzed on macrodissected (n=10) and LCM (n=5) healthy colonic, adenomatous biopsy (n=10) and LCM (n=5), macrodissected (n=10) and LCM (n=5) left-sided colorectal cancer samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes with miRWALK algorithm, miRNA expression was analyzed on colorectal cancers (n=3), adenomas (n=3) and normal tissue adjacent to tumor (NAT)(n=3) samples using the Human Panel I + II with the miRCURY Universal RT microRNA PCR protocol (Exiqon). In order to investigate the potential impact of DNA methylation on the protein levels PTGDR and SFRP1 immunohistochemistry experiments were performed. Results: A 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. COL1A2, SFRP2, SOCS3 showed hypermethylation and THBS2 showed hypomethylation both in the adenomas and tumor 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 the adenoma and tumor samples compared to the healthy colonic tissue controls that can potentially influence the expression of genes without remarkable DNA methylation alteration (e.g. BCL2, MAL, PTGS2). Decreasing protein levels of PTGDR and SFRP1 could be observed along the adenoma-carcinoma sequence. Conclusion: Genome-wide gene expression-based screening was found to be a suitable approach for the identification of genes, that can be potentially downregulated by DNA hypermethylation or miRNA upregulation. Hypermethylation of the selected markers (COL1A2, SFRP2, SOCS3) or miR21 upregulation might result in reduced expression and may contribute to the formation of colorectal cancer. Citation Format: Bela Molnar, Balint Peterfia, Alexandra Kalmar, Peter Hollosi, Zsofia Brigitta Nagy, Barnabas Wichmann, Ilona Kovalszky, Zsolt Tulassay. DNA hypermethylation or upregulated miRNA21 expression potentially leads to decreased mRNA expression of COL1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in left-sided colorectal adenoma and cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2014-422