- Email: [email protected]
593 Modeling Prostate Cancer Oncogenesis Through Developmental Alterations in Zebrafish
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
590 The DNA Methylation Landscape of Paediatric Acute Lymphoblastic Leukemia 1
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1
3
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J. Nordlund , C. Backlin ¨ , P. Wahlberg , E. Forestier , M. Heyman , 6 S. Soderh ¨ all ¨ 4 , K. Schmiegelow5 , G. Lonnerholm ¨ , M. Gustafsson2 , 1 1 A.C. Syvanen ¨ . Uppsala University, Medical Sciences Molecular Medicine, Uppsala, Sweden, 2 Uppsala University, Medical Sciences Cancer Pharmacology and Computational Medicine, Uppsala, Sweden, 3 University of ˚ Medical Biosciences, Umea, ˚ Sweden, 4 Karolinska University Hospital, Umea, Childhood Cancer Research Unit Astrid Lindgren Children’s Hospital, Stockholm, Sweden, 5 University of Copenhagen, Institute of Gynecology Obstetrics and Pediatrics, Copenhagen, Denmark, 6 Uppsala University, Women’s and Children’s Health, Uppsala, Sweden Introduction: Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, accounting for 25% of all childhood malignancies in the Nordic countries. DNA methylation plays an important role as a modulator of gene expression and in the control of cell differentiation, thus DNA methylation could be one of the factors that enhance the malignant transformation of precursor blood cells into leukemic cells. DNA methylation may also contribute to the variation between ALL patients in clinical outcome and response to treatment. Materials and Methods: Using Infinium Methylation BeadChips, we determined the methylation levels of >450,000 CpG sites in 1,002 samples, including 850 bone marrow samples collected at the time of ALL diagnosis from patients in the Nordic countries. Follow-up bone marrow samples from ALL patients taken during remission and purified CD19+, CD3+, and CD34+ cells isolated from healthy blood donors were included as controls. Four representative ALL patients were subjected to whole-genome bisulfite sequencing (BS-seq) at high coverage. Results and Discussion: The DNA methylation levels (b-values) determined with the BeadChips were highly correlated to those measured by BS-seq of DNA from the same individuals (Pearson’s R >0.95). The ALL samples displayed higher variability and a distinct overall increase in methylation levels compared to healthy blood cell populations. Over 25k of the 450k CpG sites were differentially methylated in ALL cells with a median absolute difference in methylation levels >30% compared to the controls. Of these, 79% were hypermethylated in the ALL cells and predominantly located in CpG islands and nearby the transcription start site. In contrast, the majority of the hypomethylated sites were located outside of CpG islands, in particular in CpG sites flanking islands and in gene bodies. Currently, multivariate analysis of variation in DNA methylation levels across the genome against clinical outcome is underway. Conclusion: Preliminary results show that the array-based assay and BSseq is highly reproducible. Several methylation changes that will serve as candidates for further investigation into the molecular role of DNA methylation in ALL etiology and clinical outcome of ALL patients have been identified. This study has been approved by the Nordic Society for Pediatric Hematology and Oncology. 591 Lack of MicroRNA-101 Causes E-cadherin Functional Deregulation Through EZH2 Upregulation in Intestinal Gastric Cancer J. Carvalho1 , N. van Grieken2 , P.M. Pereira3 , B. Diosdado2 , M.A.S. Santos3 , G. Meijer2 , T.E. Buffart2 , R. Seruca1 , B. Carvalho2 , C. Oliveira1 . 1 IPATIMUP, Cancer Genetics, Porto, Portugal, 2 VU University Medical Center, Department of Pathology, Amsterdam, The Netherlands, 3 University of Aveiro, RNA Biology Laboratory Department of Biology and CESAM, Aveiro, Portugal Introduction: E-cadherin expression disruption is commonly observed in metastatic epithelial cancers and a crucial step in gastric cancer (GC) initiation and progression. As aberrant expression of microRNAs often perturb the normal expression/function of pivotal cancer-related genes, we characterised and dissected a pathway that causes E-cadherin dysfunction via loss of microRNA-101 and upregulation of EZH2 expression in GC. Material and Methods: MicroRNA microarray expression profiling and ArrayCGH were used to confirm miR-101 involvement in GC and validated with quantitative real-time PCR and quantitative Snapshot genomic PCR. EZH2 and E-cadherin mRNA and protein levels were determined by real-time PCR and western-blot/co-immunofluorescence. CDH1 mutation, methylation and LOH were determined. Gain/loss of function experiments were done in KatoIII cells. E-cadherin functionality was assessed by immunofluorescence and flow cytometry. Results: We confirmed that miR-101 was significantly downregulated in GC (P < 0.0089) in comparison with normal gastric mucosas and, at least in 65% of the GC cases analysed, this downregulation was caused by deletions and/or microdeletions at miR-101 genomic loci. Moreover, around 40% of cases showing miR-101 downregulation, displayed concomitant EZH2 overexpression (at the RNA and protein levels) which, in turn, associated with loss/aberrant E-cadherin expression. Interestingly, this occurred preferentially in intestinal type GCs retaining allele(s) untargeted by classical CDH1 inactivating mechanisms. We also demonstrated that miR-101 gain of function
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or direct inhibition of EZH2 in Kato III GC cells, led to a strong depletion of endogenous EZH2 and consequent rescue of E-cadherin membranous localization, mimicking results obtained in clinical GC samples. Conclusion: In conclusion, we show for the first time that deletions and/or microdeletions at both miR-101 genomic loci cause mature miR-101 downregulation, subsequent EZH2 overexpression and E-cadherin dysfunction specifically in intestinal-type GC. 592 Are Epigenetic Mechanisms Responsible for Silencing the Calcium Sensing Receptor in Colorectal Cancer? 1 I.S. Fetahu1 , J. Hobaus ¨ , D. Hummel1 , U. Thiem1 , T. Manhardt1 , E. Kallay1 . 1 Institute of Pathophysiology and Allergy Research, Tumor Pathology, Vienna, Austria
Introduction: Epidemiological studies suggest a role for calcium in prevention of colorectal cancer (CRC). The calcium sensing receptor (CaSR) probably mediates the antiproliferative action of calcium in colon. The CaSR expression decreases during tumor progression in human CRC. We hypothesized that epigenetic mechanisms like DNA hypermethylation and histone deacetylation might be responsible for silencing the expression of the calcium sensing receptor in colorectal tumors. Material and Methods: We analyzed CaSR mRNA and protein expression in CRC tumors and cell lines by real time qRT-PCR and immunofluorescence. Bisulfite sequencing was used to determine the methylation pattern of two regions in the second promoter of the CaSR. We treated colon tumor cell lines with 5-aza-2-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, and/or Trichostatin A (TSA), a histone deacetylase inhibitor to induce the expression of the CaSR. Results and Discussion: In CRC patients we observed a significant downregulation of CaSR mRNA expression (P < 0.0001) in tumor tissues compared with the respective adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of the CaSR protein in tumors also. Bisulfite sequencing of two regions in the second promoter of the CaSR in CRC cell lines showed dense methylation of the second region. Although the methylation ranged from 2−70% among patients, there was no difference in the methylation pattern between tumor and the respective adjacent mucosa in any of the analyzed regions. Treatment with 5-aza-dC and TSA caused only modest increase of the CaSR expression, despite the presence of densely methylated CpG islands in the promoter of the CaSR. Conclusion: In our patient cohort the loss of CaSR expression in colon cancer is independent of DNA hypermethylation and histone deacetylation. 593 Modeling Prostate Cancer Oncogenesis Through Developmental Alterations in Zebrafish G. Boysen1 , C. Bourque2 , J. Huang1 , C. Barbieri1 , N. Kitabayashi1 , Z. Chen1 , F. Demichelis1 , W. Hussain1 , Y. Houvras2 , M. Rubin1 . 1 Weill Cornell Medical College, Pathology, New York, USA, 2 Weill Cornell Medical College, Surgery, New York, USA Introduction: Whole Exome and Genome sequencing has nominated the Speckle-type POZ protein (SPOP) as the most frequently mutated gene in localized prostate cancer at a frequency of up to 14% (Barbieri et al. in revision). SPOP functions as a substrate recognition subunit of a Cullin3-containing E3-ubiquitin ligase and is highly conserved among vertebrates. Although zebrafish do not have prostate glands, we explored the characterization of pathways altered as a consequence of the most frequent SPOP mutation. Zebrafish is a genetic model organism, which permits functional characterization of oncogenes and developmental pathways. The high level of conservation between zebrafish and human and the relative ease of genetic modification make zebrafish an excellent vertebrate model organism to characterize mutations found in next generation sequencing studies of human cancers. Material and Methods: Wildtype AB/ Tuebingen and p53M214K/M214K , a p53 point mutant in the DNA-binding domain were used for this study. We designed splice blocking and translation inhibiting morpholinos against Spop. Morpholinos and in vitro transcribed SPOP mRNA were injected into the yolk at the 1-cell stage. RNA library preparation and RNA-sequencing was performed. Results and Discussion: An initial analysis of Spop protein sequence revealed a high degree of conservation between zebrafish and human (98.9% amino acid identity). We performed morpholino-based knockdown of SPOP, which led to a highly reproducible phenotype characterized by malformations of brain development and overall body plan. Microinjection of human SPOP mRNA rescued this phenotype confirming the specificity of the morpholinobased knockdown. We performed RNA-sequencing on zebrafish embryos after Spop morpholino injection in order to identify pathways regulated by SPOP. These studies suggest that SPOP is involved in cell cycle regulation and activation of the DNA-damage response pathway. Furthermore, we overexpressed the most common SPOP mutant F133V in order to investigate the effect of this mutation on SPOP function. This analysis resulted in a
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european journal of cancer 48, suppl. 5 (2012) S25–S288
significantly more efficient rescue of SPOP knockdown compared to wildtype SPOP, suggesting that F133V is a gain of function mutation. Conclusion: In this study, we describe the use of zebrafish to evaluate the function of Spop in development. Our studies demonstrate that Spop is required for early organogenesis of the CNS. We identified SPOP to be a key regulator of developmental processes potentially by controlling cell cycle and DNA-damage responses. This study emphasizes the use of zebrafish as an emerging in vivo model to functionally characterize genetic alterations identified in large-scale next generation DNA-sequencing studies. We are currently analyzing transcriptome data from human prostate cancer tissue and cell lines to verify the relevance of the molecular alterations identified by Spop knockdown in zebrafish for human cancer. 596 Genetic and Epigenetic Alterations in the Embrionary Tumor Hepatoblastoma T.C. Rodrigues1 , A.C.V. Krepischi2 , M. Maschietto2 , C.M.L. Costa3 , ˜ Paulo, Genetics and D.M. Carraro2 , C. Rosenberg1 . 1 University of Sao ˜ Paulo / SP, Brazil, 2 AC Camargo Hospital, Cipe, Evolutionary Biology, Sao 3 ˜ Paulo / SP, Brazil, AC Camargo Hospital, Oncopediatrics, Sao ˜ Paulo / Sao SP, Brazil Embrionary tumors are rare malignancies originating from primary cells that have acquired somatic mutations. Hepatoblastoma (HB) is the most common liver cancer in children and adolescents, consisting of a solid embryonal tumor that presents high mortality and rapid progression. Molecular data on HB are still scarce and remain inconclusive. In the present study we were particularly interested in the delineation of somatic copy number alteration (SCNAs) and genome-wide DNA methylation patterns. We have investigated 5 HB samples. Array-CGH with a 180K oligoarray platform (OGT) was employed to detect the SCNAs, using for analysis the Nexus 6 software (Biodiscovery). The global profile of genomic imbalances showed only a few alterations in each sample (average number of SCNA per tumor = 2.8), which indicates that HBs have less genomic instability than most solid tumors. Four out of five tumors exhibited high gain of a small segment in a region previously reported as altered at 1p36.33. Total or partial aneuplody of chromosomes 2 and 20 (gains) are recurrent aberrations in HB, and they were detected in one sample. Interestingly, another tumor showed a small amplified region at 2q, narrowing the minimum chromosome segment recurrently amplified. Two HB harbored a high level gain at 5q, encompassing among others genes a miRNA. Gains in mosaicwere found at 1q, 3p and X, in addition to loss of a segment at 22q including a known cancer susceptibility gene yet not related to HB. Evaluation of the global DNA methylation pattern was performed with the 450K BeadArray platform and analyzed with the Genome Studio software (both from Illumina). A preliminary analysis disclosed 74 CpG sites significantly more methylated in tumors than in normal liver, nearly half of which lying in genes involved in metabolic processes. In complementary, we detected 34 demethylated CpGs in HB samples, affecting genes involved mainly in cell fate commitment e epithelium development. The group of HBs investigated will be expanded to contribute to elucidation on which alterations in genes and molecular pathways lead to impairment of normal liver differentiation and subsequent oncogenic process. Financial support: CAPES, FAPESP. 597 Using an in Vitro Model of Epithelial-Mesenchymal-Epithelial Transitions to Uncover Novel Biological Mechanisms P. Oliveira1 , J. Carvalho1 , M. Azevedo1 , A. Moussavi2 , S.S. Pinho3 , 4 , J. Lima4 , C. Reis3 , D.G. Huntsman2 , C. Oliveira1 . 1 IPATIMUP, V. Maximo ´ Cancer Genetics, Porto, Portugal, 2 Vancouver General Hospital and British Columbia Cancer Agency, Department of Pathology, Vancouver, Canada, 3 IPATIMUP, Carcinogenesis, Porto, Portugal, 4 IPATIMUP, Cancer Biology, Porto, Portugal Background: Epithelial-mesenchymal transition (EMT) and mesenchymalepithelial transition (MET) are fundamental mechanisms controlling multiple events during embryonic development and cancer. Cancerous cells undergoing EMT, exhibit a mesenchymal-like phenotype with concomitant polarity loss, increased invasibility and apoptosis resistance, features predicted to enable metastization. The establishment of cancerous cells at novel locations is a key event in metastization and only possible due to expression plasticity characteristic of cells that are able to undergo MET. Many molecules have been proved to induce EMT and others to be modulated by EMT. However, never was the variation of whole transcriptome been assessed during these crucial biological processes. Therefore our aim was to assess the whole transcriptome variations in an in vitro model of EMT and MET, in order to clarify major biological changes underlying these processes. Materials and Methods: We have reproduced EMT and MET in vitro by treating a normal mouse mammary cell line (EpH4) with/without TGF-b1. DNAse treated total RNA extracted at distinct EMT/MET-timepoints was then subjected to whole transcriptome sequencing (Illumina GA). Bioinformatic
Sunday 8 − Tuesday 10 July 2012
analysis was performed using in house pipelines and commercially available software (Ingenuity Systems Pathway Analysis Software and Database for Annotation, Visualization and Integrated Discovery Software). RNA expression alterations were verified by qRT-PCR, protein expression by Western Blot, immunoprecipitation and immunofluorescence and DNA methylation alterations by bisulfite treatment. Metabolism intermediates were measured using ELISA. Results: We confirmed the occurrence of EMT and MET via analysis of the differential transcription of classical epithelial and mesenchymal markers. In our in vitro EMT/MET model, we have found several thousands of genes differentially expressed and our bioinformatics analysis correlated this differential activation with uncounted cancer and metastasis related pathways. Moreover, we have uncovered novel biological mechanisms underlying EMT/MET such as differential glycosylation of E-cadherin, alternative activation of metabolic pathways and novel epigenetic mechanisms, underlying activation/repression of recently annotated genes. Conclusions: We were able to establish a dynamic in vitro model of EMT/MET, which has enable us to uncover novel biological mechanisms using a nonbiased genome wide approach. 598 Follicular Thyroid Cancer Molecular Markers Validation in FFPE Material B. Wojtas1 , A. Pfeifer1 , T. Stokowy1 , M. Eszlinger2 , M. Jarzab3 , A. Czarniecka4 , S. Hauptmann5 , E. Stobiecka6 , R. Paschke2 , B. Jarzab1 . 1 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Nuclear Medicine and Oncologic Endocrinology, Gliwice, Poland, 2 University of Leipzig, Division of Endocrinology and Nephrology, Leipzig, Germany, 3 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Radiotherapy and Chemotherapy, Gliwice, Poland, 4 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Oncological Surgery, Gliwice, Poland, 5 University of Halle, Institute for Pathology, Halle, Germany, 6 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Tumor Pathology, Gliwice, Poland Introduction: Follicular thyroid cancer (FTC) is diagnostically challenging as its cellular morphology is similar to a follicular thyroid adenoma (FTA). In the last decade many gene expression studies were performed to improve a relatively high misclassification rate in routine histopathological diagnosis. One of the most interesting studies was Borup et al. 2010 that is the largest follicular thyroid tumour dataset published so far. Materials and Methods: Gene expression profile was carried out in 52 samples (25 follicular carcinomas and 27 adenomas) by HG-U133 Plus 2.0 oligonucleotide microarrays (Affymetrix). Whole dataset was analysed by Singular Value Decomposition to identify the major sources of variability. In the next step, a subset of our well-defined samples (concordance of diagnosis between two experts in thyroid pathology) and samples from Borup et al. were combined in one powerful supervised analysis to look for the most important differentially expressed genes. Subsequently 8 transcripts, that came as a result of the analysis were validated by quantitative PCR using formalin-fixed paraffin-embedded (FFPE) material. Results and Discussion: Immunologic response was the strongest supergene discriminating FTC and FTA in unsupervised analysis (it accounted for 14.5% of the total variance). The second important source of variability was the proliferation rate of thyroid cells. 5 of 8 genes selected from supervised analysis (ELMO1, EMCN, ITIH5, KCNAB1, SLCO2A1) could be amplified by quantitative real-time PCR in FFPE material in the independent set of samples (N = 71). Validated genes were all significantly (p < 0.05) down-regulated in FTC when compared to FTA. The 5-gene classifier build on genes described above had a sensitivity of 71% and specificity 72%, when tested on QPCR dataset. The classifier was also tested in some available external microarray datasets, where it showed very good performance. Conclusion: ELMO1, EMCN, ITIH5, KCNAB1 and SLCO2A1 are genes important for follicular thyroid malignancy development and information about their level can be helpful in FTC diagnosis. This work was supported by Ministry of Science and Higher Education grant nr N401 072637 and N N403 194340 and by Foundation of Polish Science International PhD Projects (MPD) program. 599 Up-regulation of ER-b, p53 and p21 Sensitizes Tamoxifen Resistant Breast Cancer Cells to Tamoxifen C.A. Pitta1 , P. Papageorgis1 , A.I. Constantinou1 . 1 University of Cyprus, Biological Sciences, Lefkosia (Nicosia), Cyprus Breast tumors expressing estrogen receptors (ER) a and b respond well to therapeutic and preventative strategies with Selective Estrogen Receptor Modulators (SERMs) such as tamoxifen. However, a number of breast cancers are either resistant or develop resistance to tamoxifen and other SERMs. The promoters of ER-a and ER-b are rich in CpG islands making them susceptible to epigenetic modifications. Methylated CpG clusters are known to
european journal of cancer 48, suppl. 5 (2012) S25–S288
590 The DNA Methylation Landscape of Paediatric Acute Lymphoblastic Leukemia 1
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J. Nordlund , C. Backlin ¨ , P. Wahlberg , E. Forestier , M. Heyman , 6 S. Soderh ¨ all ¨ 4 , K. Schmiegelow5 , G. Lonnerholm ¨ , M. Gustafsson2 , 1 1 A.C. Syvanen ¨ . Uppsala University, Medical Sciences Molecular Medicine, Uppsala, Sweden, 2 Uppsala University, Medical Sciences Cancer Pharmacology and Computational Medicine, Uppsala, Sweden, 3 University of ˚ Medical Biosciences, Umea, ˚ Sweden, 4 Karolinska University Hospital, Umea, Childhood Cancer Research Unit Astrid Lindgren Children’s Hospital, Stockholm, Sweden, 5 University of Copenhagen, Institute of Gynecology Obstetrics and Pediatrics, Copenhagen, Denmark, 6 Uppsala University, Women’s and Children’s Health, Uppsala, Sweden Introduction: Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, accounting for 25% of all childhood malignancies in the Nordic countries. DNA methylation plays an important role as a modulator of gene expression and in the control of cell differentiation, thus DNA methylation could be one of the factors that enhance the malignant transformation of precursor blood cells into leukemic cells. DNA methylation may also contribute to the variation between ALL patients in clinical outcome and response to treatment. Materials and Methods: Using Infinium Methylation BeadChips, we determined the methylation levels of >450,000 CpG sites in 1,002 samples, including 850 bone marrow samples collected at the time of ALL diagnosis from patients in the Nordic countries. Follow-up bone marrow samples from ALL patients taken during remission and purified CD19+, CD3+, and CD34+ cells isolated from healthy blood donors were included as controls. Four representative ALL patients were subjected to whole-genome bisulfite sequencing (BS-seq) at high coverage. Results and Discussion: The DNA methylation levels (b-values) determined with the BeadChips were highly correlated to those measured by BS-seq of DNA from the same individuals (Pearson’s R >0.95). The ALL samples displayed higher variability and a distinct overall increase in methylation levels compared to healthy blood cell populations. Over 25k of the 450k CpG sites were differentially methylated in ALL cells with a median absolute difference in methylation levels >30% compared to the controls. Of these, 79% were hypermethylated in the ALL cells and predominantly located in CpG islands and nearby the transcription start site. In contrast, the majority of the hypomethylated sites were located outside of CpG islands, in particular in CpG sites flanking islands and in gene bodies. Currently, multivariate analysis of variation in DNA methylation levels across the genome against clinical outcome is underway. Conclusion: Preliminary results show that the array-based assay and BSseq is highly reproducible. Several methylation changes that will serve as candidates for further investigation into the molecular role of DNA methylation in ALL etiology and clinical outcome of ALL patients have been identified. This study has been approved by the Nordic Society for Pediatric Hematology and Oncology. 591 Lack of MicroRNA-101 Causes E-cadherin Functional Deregulation Through EZH2 Upregulation in Intestinal Gastric Cancer J. Carvalho1 , N. van Grieken2 , P.M. Pereira3 , B. Diosdado2 , M.A.S. Santos3 , G. Meijer2 , T.E. Buffart2 , R. Seruca1 , B. Carvalho2 , C. Oliveira1 . 1 IPATIMUP, Cancer Genetics, Porto, Portugal, 2 VU University Medical Center, Department of Pathology, Amsterdam, The Netherlands, 3 University of Aveiro, RNA Biology Laboratory Department of Biology and CESAM, Aveiro, Portugal Introduction: E-cadherin expression disruption is commonly observed in metastatic epithelial cancers and a crucial step in gastric cancer (GC) initiation and progression. As aberrant expression of microRNAs often perturb the normal expression/function of pivotal cancer-related genes, we characterised and dissected a pathway that causes E-cadherin dysfunction via loss of microRNA-101 and upregulation of EZH2 expression in GC. Material and Methods: MicroRNA microarray expression profiling and ArrayCGH were used to confirm miR-101 involvement in GC and validated with quantitative real-time PCR and quantitative Snapshot genomic PCR. EZH2 and E-cadherin mRNA and protein levels were determined by real-time PCR and western-blot/co-immunofluorescence. CDH1 mutation, methylation and LOH were determined. Gain/loss of function experiments were done in KatoIII cells. E-cadherin functionality was assessed by immunofluorescence and flow cytometry. Results: We confirmed that miR-101 was significantly downregulated in GC (P < 0.0089) in comparison with normal gastric mucosas and, at least in 65% of the GC cases analysed, this downregulation was caused by deletions and/or microdeletions at miR-101 genomic loci. Moreover, around 40% of cases showing miR-101 downregulation, displayed concomitant EZH2 overexpression (at the RNA and protein levels) which, in turn, associated with loss/aberrant E-cadherin expression. Interestingly, this occurred preferentially in intestinal type GCs retaining allele(s) untargeted by classical CDH1 inactivating mechanisms. We also demonstrated that miR-101 gain of function
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or direct inhibition of EZH2 in Kato III GC cells, led to a strong depletion of endogenous EZH2 and consequent rescue of E-cadherin membranous localization, mimicking results obtained in clinical GC samples. Conclusion: In conclusion, we show for the first time that deletions and/or microdeletions at both miR-101 genomic loci cause mature miR-101 downregulation, subsequent EZH2 overexpression and E-cadherin dysfunction specifically in intestinal-type GC. 592 Are Epigenetic Mechanisms Responsible for Silencing the Calcium Sensing Receptor in Colorectal Cancer? 1 I.S. Fetahu1 , J. Hobaus ¨ , D. Hummel1 , U. Thiem1 , T. Manhardt1 , E. Kallay1 . 1 Institute of Pathophysiology and Allergy Research, Tumor Pathology, Vienna, Austria
Introduction: Epidemiological studies suggest a role for calcium in prevention of colorectal cancer (CRC). The calcium sensing receptor (CaSR) probably mediates the antiproliferative action of calcium in colon. The CaSR expression decreases during tumor progression in human CRC. We hypothesized that epigenetic mechanisms like DNA hypermethylation and histone deacetylation might be responsible for silencing the expression of the calcium sensing receptor in colorectal tumors. Material and Methods: We analyzed CaSR mRNA and protein expression in CRC tumors and cell lines by real time qRT-PCR and immunofluorescence. Bisulfite sequencing was used to determine the methylation pattern of two regions in the second promoter of the CaSR. We treated colon tumor cell lines with 5-aza-2-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, and/or Trichostatin A (TSA), a histone deacetylase inhibitor to induce the expression of the CaSR. Results and Discussion: In CRC patients we observed a significant downregulation of CaSR mRNA expression (P < 0.0001) in tumor tissues compared with the respective adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of the CaSR protein in tumors also. Bisulfite sequencing of two regions in the second promoter of the CaSR in CRC cell lines showed dense methylation of the second region. Although the methylation ranged from 2−70% among patients, there was no difference in the methylation pattern between tumor and the respective adjacent mucosa in any of the analyzed regions. Treatment with 5-aza-dC and TSA caused only modest increase of the CaSR expression, despite the presence of densely methylated CpG islands in the promoter of the CaSR. Conclusion: In our patient cohort the loss of CaSR expression in colon cancer is independent of DNA hypermethylation and histone deacetylation. 593 Modeling Prostate Cancer Oncogenesis Through Developmental Alterations in Zebrafish G. Boysen1 , C. Bourque2 , J. Huang1 , C. Barbieri1 , N. Kitabayashi1 , Z. Chen1 , F. Demichelis1 , W. Hussain1 , Y. Houvras2 , M. Rubin1 . 1 Weill Cornell Medical College, Pathology, New York, USA, 2 Weill Cornell Medical College, Surgery, New York, USA Introduction: Whole Exome and Genome sequencing has nominated the Speckle-type POZ protein (SPOP) as the most frequently mutated gene in localized prostate cancer at a frequency of up to 14% (Barbieri et al. in revision). SPOP functions as a substrate recognition subunit of a Cullin3-containing E3-ubiquitin ligase and is highly conserved among vertebrates. Although zebrafish do not have prostate glands, we explored the characterization of pathways altered as a consequence of the most frequent SPOP mutation. Zebrafish is a genetic model organism, which permits functional characterization of oncogenes and developmental pathways. The high level of conservation between zebrafish and human and the relative ease of genetic modification make zebrafish an excellent vertebrate model organism to characterize mutations found in next generation sequencing studies of human cancers. Material and Methods: Wildtype AB/ Tuebingen and p53M214K/M214K , a p53 point mutant in the DNA-binding domain were used for this study. We designed splice blocking and translation inhibiting morpholinos against Spop. Morpholinos and in vitro transcribed SPOP mRNA were injected into the yolk at the 1-cell stage. RNA library preparation and RNA-sequencing was performed. Results and Discussion: An initial analysis of Spop protein sequence revealed a high degree of conservation between zebrafish and human (98.9% amino acid identity). We performed morpholino-based knockdown of SPOP, which led to a highly reproducible phenotype characterized by malformations of brain development and overall body plan. Microinjection of human SPOP mRNA rescued this phenotype confirming the specificity of the morpholinobased knockdown. We performed RNA-sequencing on zebrafish embryos after Spop morpholino injection in order to identify pathways regulated by SPOP. These studies suggest that SPOP is involved in cell cycle regulation and activation of the DNA-damage response pathway. Furthermore, we overexpressed the most common SPOP mutant F133V in order to investigate the effect of this mutation on SPOP function. This analysis resulted in a
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european journal of cancer 48, suppl. 5 (2012) S25–S288
significantly more efficient rescue of SPOP knockdown compared to wildtype SPOP, suggesting that F133V is a gain of function mutation. Conclusion: In this study, we describe the use of zebrafish to evaluate the function of Spop in development. Our studies demonstrate that Spop is required for early organogenesis of the CNS. We identified SPOP to be a key regulator of developmental processes potentially by controlling cell cycle and DNA-damage responses. This study emphasizes the use of zebrafish as an emerging in vivo model to functionally characterize genetic alterations identified in large-scale next generation DNA-sequencing studies. We are currently analyzing transcriptome data from human prostate cancer tissue and cell lines to verify the relevance of the molecular alterations identified by Spop knockdown in zebrafish for human cancer. 596 Genetic and Epigenetic Alterations in the Embrionary Tumor Hepatoblastoma T.C. Rodrigues1 , A.C.V. Krepischi2 , M. Maschietto2 , C.M.L. Costa3 , ˜ Paulo, Genetics and D.M. Carraro2 , C. Rosenberg1 . 1 University of Sao ˜ Paulo / SP, Brazil, 2 AC Camargo Hospital, Cipe, Evolutionary Biology, Sao 3 ˜ Paulo / SP, Brazil, AC Camargo Hospital, Oncopediatrics, Sao ˜ Paulo / Sao SP, Brazil Embrionary tumors are rare malignancies originating from primary cells that have acquired somatic mutations. Hepatoblastoma (HB) is the most common liver cancer in children and adolescents, consisting of a solid embryonal tumor that presents high mortality and rapid progression. Molecular data on HB are still scarce and remain inconclusive. In the present study we were particularly interested in the delineation of somatic copy number alteration (SCNAs) and genome-wide DNA methylation patterns. We have investigated 5 HB samples. Array-CGH with a 180K oligoarray platform (OGT) was employed to detect the SCNAs, using for analysis the Nexus 6 software (Biodiscovery). The global profile of genomic imbalances showed only a few alterations in each sample (average number of SCNA per tumor = 2.8), which indicates that HBs have less genomic instability than most solid tumors. Four out of five tumors exhibited high gain of a small segment in a region previously reported as altered at 1p36.33. Total or partial aneuplody of chromosomes 2 and 20 (gains) are recurrent aberrations in HB, and they were detected in one sample. Interestingly, another tumor showed a small amplified region at 2q, narrowing the minimum chromosome segment recurrently amplified. Two HB harbored a high level gain at 5q, encompassing among others genes a miRNA. Gains in mosaicwere found at 1q, 3p and X, in addition to loss of a segment at 22q including a known cancer susceptibility gene yet not related to HB. Evaluation of the global DNA methylation pattern was performed with the 450K BeadArray platform and analyzed with the Genome Studio software (both from Illumina). A preliminary analysis disclosed 74 CpG sites significantly more methylated in tumors than in normal liver, nearly half of which lying in genes involved in metabolic processes. In complementary, we detected 34 demethylated CpGs in HB samples, affecting genes involved mainly in cell fate commitment e epithelium development. The group of HBs investigated will be expanded to contribute to elucidation on which alterations in genes and molecular pathways lead to impairment of normal liver differentiation and subsequent oncogenic process. Financial support: CAPES, FAPESP. 597 Using an in Vitro Model of Epithelial-Mesenchymal-Epithelial Transitions to Uncover Novel Biological Mechanisms P. Oliveira1 , J. Carvalho1 , M. Azevedo1 , A. Moussavi2 , S.S. Pinho3 , 4 , J. Lima4 , C. Reis3 , D.G. Huntsman2 , C. Oliveira1 . 1 IPATIMUP, V. Maximo ´ Cancer Genetics, Porto, Portugal, 2 Vancouver General Hospital and British Columbia Cancer Agency, Department of Pathology, Vancouver, Canada, 3 IPATIMUP, Carcinogenesis, Porto, Portugal, 4 IPATIMUP, Cancer Biology, Porto, Portugal Background: Epithelial-mesenchymal transition (EMT) and mesenchymalepithelial transition (MET) are fundamental mechanisms controlling multiple events during embryonic development and cancer. Cancerous cells undergoing EMT, exhibit a mesenchymal-like phenotype with concomitant polarity loss, increased invasibility and apoptosis resistance, features predicted to enable metastization. The establishment of cancerous cells at novel locations is a key event in metastization and only possible due to expression plasticity characteristic of cells that are able to undergo MET. Many molecules have been proved to induce EMT and others to be modulated by EMT. However, never was the variation of whole transcriptome been assessed during these crucial biological processes. Therefore our aim was to assess the whole transcriptome variations in an in vitro model of EMT and MET, in order to clarify major biological changes underlying these processes. Materials and Methods: We have reproduced EMT and MET in vitro by treating a normal mouse mammary cell line (EpH4) with/without TGF-b1. DNAse treated total RNA extracted at distinct EMT/MET-timepoints was then subjected to whole transcriptome sequencing (Illumina GA). Bioinformatic
Sunday 8 − Tuesday 10 July 2012
analysis was performed using in house pipelines and commercially available software (Ingenuity Systems Pathway Analysis Software and Database for Annotation, Visualization and Integrated Discovery Software). RNA expression alterations were verified by qRT-PCR, protein expression by Western Blot, immunoprecipitation and immunofluorescence and DNA methylation alterations by bisulfite treatment. Metabolism intermediates were measured using ELISA. Results: We confirmed the occurrence of EMT and MET via analysis of the differential transcription of classical epithelial and mesenchymal markers. In our in vitro EMT/MET model, we have found several thousands of genes differentially expressed and our bioinformatics analysis correlated this differential activation with uncounted cancer and metastasis related pathways. Moreover, we have uncovered novel biological mechanisms underlying EMT/MET such as differential glycosylation of E-cadherin, alternative activation of metabolic pathways and novel epigenetic mechanisms, underlying activation/repression of recently annotated genes. Conclusions: We were able to establish a dynamic in vitro model of EMT/MET, which has enable us to uncover novel biological mechanisms using a nonbiased genome wide approach. 598 Follicular Thyroid Cancer Molecular Markers Validation in FFPE Material B. Wojtas1 , A. Pfeifer1 , T. Stokowy1 , M. Eszlinger2 , M. Jarzab3 , A. Czarniecka4 , S. Hauptmann5 , E. Stobiecka6 , R. Paschke2 , B. Jarzab1 . 1 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Nuclear Medicine and Oncologic Endocrinology, Gliwice, Poland, 2 University of Leipzig, Division of Endocrinology and Nephrology, Leipzig, Germany, 3 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Radiotherapy and Chemotherapy, Gliwice, Poland, 4 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Oncological Surgery, Gliwice, Poland, 5 University of Halle, Institute for Pathology, Halle, Germany, 6 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Tumor Pathology, Gliwice, Poland Introduction: Follicular thyroid cancer (FTC) is diagnostically challenging as its cellular morphology is similar to a follicular thyroid adenoma (FTA). In the last decade many gene expression studies were performed to improve a relatively high misclassification rate in routine histopathological diagnosis. One of the most interesting studies was Borup et al. 2010 that is the largest follicular thyroid tumour dataset published so far. Materials and Methods: Gene expression profile was carried out in 52 samples (25 follicular carcinomas and 27 adenomas) by HG-U133 Plus 2.0 oligonucleotide microarrays (Affymetrix). Whole dataset was analysed by Singular Value Decomposition to identify the major sources of variability. In the next step, a subset of our well-defined samples (concordance of diagnosis between two experts in thyroid pathology) and samples from Borup et al. were combined in one powerful supervised analysis to look for the most important differentially expressed genes. Subsequently 8 transcripts, that came as a result of the analysis were validated by quantitative PCR using formalin-fixed paraffin-embedded (FFPE) material. Results and Discussion: Immunologic response was the strongest supergene discriminating FTC and FTA in unsupervised analysis (it accounted for 14.5% of the total variance). The second important source of variability was the proliferation rate of thyroid cells. 5 of 8 genes selected from supervised analysis (ELMO1, EMCN, ITIH5, KCNAB1, SLCO2A1) could be amplified by quantitative real-time PCR in FFPE material in the independent set of samples (N = 71). Validated genes were all significantly (p < 0.05) down-regulated in FTC when compared to FTA. The 5-gene classifier build on genes described above had a sensitivity of 71% and specificity 72%, when tested on QPCR dataset. The classifier was also tested in some available external microarray datasets, where it showed very good performance. Conclusion: ELMO1, EMCN, ITIH5, KCNAB1 and SLCO2A1 are genes important for follicular thyroid malignancy development and information about their level can be helpful in FTC diagnosis. This work was supported by Ministry of Science and Higher Education grant nr N401 072637 and N N403 194340 and by Foundation of Polish Science International PhD Projects (MPD) program. 599 Up-regulation of ER-b, p53 and p21 Sensitizes Tamoxifen Resistant Breast Cancer Cells to Tamoxifen C.A. Pitta1 , P. Papageorgis1 , A.I. Constantinou1 . 1 University of Cyprus, Biological Sciences, Lefkosia (Nicosia), Cyprus Breast tumors expressing estrogen receptors (ER) a and b respond well to therapeutic and preventative strategies with Selective Estrogen Receptor Modulators (SERMs) such as tamoxifen. However, a number of breast cancers are either resistant or develop resistance to tamoxifen and other SERMs. The promoters of ER-a and ER-b are rich in CpG islands making them susceptible to epigenetic modifications. Methylated CpG clusters are known to