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597 Using an in Vitro Model of Epithelial-Mesenchymal-Epithelial Transitions to Uncover Novel Biological Mechanisms
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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
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