- Email: [email protected]
599 Up-regulation of ER-b, p53 and p21 Sensitizes Tamoxifen Resistant Breast Cancer Cells to Tamoxifen
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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
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
be associated with gene silencing. Our hypothesis is that tamoxifen resistant breast cancer cells can be sensitized to tamoxifen after treatment with either a demethylating agent (such as 5-AZA) or a histone deacetylase inhibitor (such as trichostatin, TSA) or their combination. These treatments are expected to activate the transcription of ER-a and ER-b m-RNA and produce functional protein reversing in this manner tamoxifen resistance. Using the MTT assay we determined the growth rates of MCF-7, MCF-7/TAM-R and MDA-MB-231 in the presence of 5-AZA, TSA and their combinations in the presence or absence of 4-OH tamoxifen. We identified the conditions (i.e order of inhibitor addition and concentration) that reverse tamoxifen resistance and those providing the maximum cell death of the MCF-7 and MCF-7/TAM-R. We found that pretreatment with 5-AZA (2 mM) or TSA (0.1 mM) enabled tamoxifen (10 mM) to produce a considerable cell death (over 50%). Interestingly, MCF-7/TAM-R cells that normally do not express ER-b, after 24 hours of treatment with TSA and 5-AZA produced a marked increased in the expression of ER-b as determined by both Western Blot and fluorescence microscopy. Following 48 hours of treatment the levels of ER-b were further increased. Additionally, the combination of 5-AZA and TSA increased the levels of pS2 by 10-fold compared to TSA alone and it was not further increased by the addition of 4-OH tamoxifen. Moreover, following 48 hours treatment with 5-AZA/TSA and tamoxifen the levels of p21 and p53 were substantially increased compared with the vehicle control. These results show: (a) the reversal of tamoxifen resistance can be achieved with relatively low (non-toxic) concentrations of 5-AZA/TSA, and (b) the upregulation of ER-b, p53 and p21 are essential components of the molecular pathway leading to this reversal. These findings may find applications in clinical therapeutic protocols in the treatment of tamoxifen resistant breast cancer. 600 Agreement Between SNP6.0 and Cytogenetic 2.7M Whole-genome Microarrays From Affymetrix in DLBCL J. Bodker1 , P. Johansen2 , C. Gyrup3 , A. Schmitz1 , P. Jensen1 , H. Johnsen1 , M. Bøgsted1 , K. Dybkær1 , M. Nyegaard1 . 1 Haematological Research Laboratory, Department of Haematology Aalborg Hospital Aarhus University Hospital, Aalborg, Denmark, 2 Department of Pathology Aalborg Hospital Aarhus University Hospital, Aalborg, Denmark, 3 Department of Biotechnology Chemistry and Environmental Engineering Aalborg University, Aalborg, Denmark Introduction: The Affymetrix SNP6.0 genome wide array is commonly used in research for detection of numeric genomic alterations in many haematological malignancies and has been found of prognostic impact. In 2009 Affymetrix launched the cytogenetic 2.7M whole-genome array (Cyto2.7M), which only required 100 ng of genomic DNA (five times less than the SNP6.0 array). Processing time was reduced approximately four times to one day and increased coverage in cancer related genes. The Cyto2.7M array performance on multiple cancer samples has not previously been reported. Materials and Method: We compared the SNP6.0 and the Cyto2.7M types of array on eight cancer samples of diffuse large B-cell lymphoma (DLBCL), known to associate with a large number of deletions and amplifications. Furthermore we evaluated the copy number of selected regions by qPCR. Cyto2.7M arrays were performed on 80 Danish DLBCL’s. Results and Discussion: In the comparison analysis, we identified 226 copy number variations (CNV) of chromosomal gain (42.5%) and loss (57.5%) and of these 199 was present in both array types. To evaluate the CNV boundaries, we calculated the delta break point (DBP) of identified segments, and in 91% of cases the DBP was below 200 kb − the detection limit set in this comparison. The copy number calls of CNVs was analyzed using the smooth signal, and was in all, but 26 events, identical. To determine the actual copy number state in these 26 regions, 6 of these were selected and evaluated by qPCR. Presence of loss of heterozygosity was evaluated for large regions (>10 Mb), and full consistency between arrays was observed. The information obtained from these microarrays can complement data from FISH analysis, as the actual copy number state of a given region is identified; yet the precise translocation is not identified. The occurrence of LOH could suggest the presence of a tumour suppressor gene, although this will have to be validated through further studies. The cytogenetic status of the 80 DLBCL samples were evaluated and compared to published results from Bea et al. 2005. Conclusion: The high degree of consistency between the array types suggests that the Cyto2.7M array is well suited to detect genomic aberrations for cancer samples, and because of the lower input requirements, this could enable genomic analysis of samples where only limited DNA is available. Furthermore, the Cyto2.7M array confirmed the cytogenetic background of DLBCL previously published.
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602 Frequent Epigenetic Inactivation of KEAP1 Gene in Breast Cancer R. Barbano1 , B. Pasculli1 , L.A. Muscarella1 , A. La Torre1 , D. Trombetta1 , R. Murgo2 , V.M. Valori3 , E. Maiello3 , V.M. Fazio4 , P. Parrella4 . 1 IRCCS Casa Sollievo Della Sofferenza, Oncology Laboratory, San Giovanni Rotondo(FG), Italy, 2 IRCCS Casa Sollievo Della Sofferenza, Breast Unit, San Giovanni Rotondo(FG), Italy, 3 IRCCS Casa Sollievo Della Sofferenza, Department of Oncology, San Giovanni Rotondo(FG), Italy, 4 IRCCS Casa Sollievo Della Sofferenza, Laboratory of Oncology, San Giovanni Rotondo(FG), Italy Background: The Nrf2/Keap1 pathway is a master regulator of several redox-sensitive genes implicated in resistance of tumour cells against chemotherapeutic drugs. Recent data suggest that epigenetic mechanisms may play a pivotal role in the regulation of KEAP1 expression. Material and Methods: We determined KEAP1 promoter methylation status in 50 breast cancers (BC), and 6 normal breast tissues (NBT) obtained from reductive mammoplasty. Normal breast tissue adjacent to tumour (NBAT) was available for 45 of the 50 breast cancer cases. In ten cases paired Atypical Ductal Hyperplasia (ADH) and/or Ductal Carcinoma in situ (DCIS) lesions were available along with NBT and tumour tissues. Promoter methylation analysis was performed using a quantitative methylation specific PCR assay in real time (QMSP). Results: Methylation levels were significantly higher in BC (Median 2.3, IQR 0– 17.05) as compared with NBT (Median 0, IQR 0−0) (P = 0.0001 Mann Whitney Test). Overall methylation at the KEAP1 promoter region was detected in 38 out of the 50 BC (76%). Similar level of methylation were detected in normal breast tissues adjacent to tumour (Median 5.27, IQR 0–17.05), ADH (Median 3.85, IQR 1.57–38.46) and DCIS (Median 2.23, IQR 1.27–7.66). Significant different levels of promoter methylation were demonstrated in the three breast cancer subgroups characterized by steroid receptor status and HER2 amplification (triple negative, estrogen receptor positive, and HER2 amplified tumours) (P = 0.04, Kruskall Wallis Test). Conclusions: Our results suggest that deregulation of the Nrf2/Keap1 system could play a pivotal role in the cancerogenesis of breast cancer. Studies are in progress to determine whether KEAP1 abnormalities may contribute to disease progression prediction and response to therapy in these patients. 603 The Effect of p53 on the DNA Repair Enzyme Thymine DNA Glycosylase N. Meireles da Costa1 , A. Hautefeuille2 , M.P. Cros3 , M.E. Melendez4 , P. Swann5 , P. Hainaut2 , L.F. Ribeiro Pinto1 . 1 Instituto Nacional do ˆ ˜ de Medicina Experimental, Rio de Janeiro, Cancer (INCA), Divisao Brazil, 2 International Agency for Research on Cancer (IARC), Molecular Carcinogenesis Group, Lyon, France, 3 International Agency for Research on Cancer (IARC), Epigenetics Group, Lyon, France Background: Thymine DNA glycosylase (TDG) belongs to the superfamily of the uracil DNA glycosylases and is the first enzyme in the base excision repair pathway that removes thymine from the G·T mismatches at CpG sites. Besides its antimutagenic role in DNA repair, the versatile TDG plays a crucial role in maintaining appropriate epigenetic regulation by interacting with transcription factors and histone acetylases, and by acting in the demethylation process of genes previously silenced by promoter methylation and hence reactivating them. Therefore the mechanisms by which proper functioning of TDG is assured may be critical for genetic and epigenetic stability. The tumor supressor TP53 has essential roles in maintaining genomic stability. p53 exerts its control over several DNA-repair pathways, either directly or indirectly. However, so far, there is no information on whether p53 may control TDG. Materials and Methods: Different cell lines mutated in TP53 (MN1 and MDD2 − isogenic breast cancer cell lines harboring either wild-type or inactive p53, respectively; TE-13 and TE-1 − esophageal squamous cell carcinoma cell lines lacking p53 or presenting a temperature-sensitive p53 mutant, respectively) and a normal human bronchial epithelial cell line (NHBE) were used. TP53 was inhibited by using small interfering (si) RNA; increase of p53 levels was induced by transfecting a p53 expression vector; DNA damage was achieved by treating cells with either doxorubicin or MMS. Levels of TDG mRNA and protein expression were analysed after treatments by Quantitative − RT PCR and Western blotting, respectively. Binding of p53 to TDG promoter region was assessed by Chromatin Immunoprecipitation (ChIP) assay and activation of TDG promoter by luciferase assay. Methylation analysis of 6 different gene promoters were performed in samples from 10 Li-Fraumeni TP53 mutation carriers subjects and 10 non-carriers subjects by using pyrosequencing. Results and Discussion: In this study we show that wild-type p53 transcriptionally regulates TDG expression. ChIP and luciferase assays revealed that wild-type p53 binds to a TDG promoter domain containing two p53 consensus response elements (p53RE), inducing its transcription. Using a variety of cell lines we demonstrate that TDG mRNA and protein expression levels are induced by DNA damage in a p53-dependent manner. Although not statistically significant, methylation levels of the analysed gene promoters were found slightly decrease in Li-Fraumeni p53 mutation carriers subjects,
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
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
be associated with gene silencing. Our hypothesis is that tamoxifen resistant breast cancer cells can be sensitized to tamoxifen after treatment with either a demethylating agent (such as 5-AZA) or a histone deacetylase inhibitor (such as trichostatin, TSA) or their combination. These treatments are expected to activate the transcription of ER-a and ER-b m-RNA and produce functional protein reversing in this manner tamoxifen resistance. Using the MTT assay we determined the growth rates of MCF-7, MCF-7/TAM-R and MDA-MB-231 in the presence of 5-AZA, TSA and their combinations in the presence or absence of 4-OH tamoxifen. We identified the conditions (i.e order of inhibitor addition and concentration) that reverse tamoxifen resistance and those providing the maximum cell death of the MCF-7 and MCF-7/TAM-R. We found that pretreatment with 5-AZA (2 mM) or TSA (0.1 mM) enabled tamoxifen (10 mM) to produce a considerable cell death (over 50%). Interestingly, MCF-7/TAM-R cells that normally do not express ER-b, after 24 hours of treatment with TSA and 5-AZA produced a marked increased in the expression of ER-b as determined by both Western Blot and fluorescence microscopy. Following 48 hours of treatment the levels of ER-b were further increased. Additionally, the combination of 5-AZA and TSA increased the levels of pS2 by 10-fold compared to TSA alone and it was not further increased by the addition of 4-OH tamoxifen. Moreover, following 48 hours treatment with 5-AZA/TSA and tamoxifen the levels of p21 and p53 were substantially increased compared with the vehicle control. These results show: (a) the reversal of tamoxifen resistance can be achieved with relatively low (non-toxic) concentrations of 5-AZA/TSA, and (b) the upregulation of ER-b, p53 and p21 are essential components of the molecular pathway leading to this reversal. These findings may find applications in clinical therapeutic protocols in the treatment of tamoxifen resistant breast cancer. 600 Agreement Between SNP6.0 and Cytogenetic 2.7M Whole-genome Microarrays From Affymetrix in DLBCL J. Bodker1 , P. Johansen2 , C. Gyrup3 , A. Schmitz1 , P. Jensen1 , H. Johnsen1 , M. Bøgsted1 , K. Dybkær1 , M. Nyegaard1 . 1 Haematological Research Laboratory, Department of Haematology Aalborg Hospital Aarhus University Hospital, Aalborg, Denmark, 2 Department of Pathology Aalborg Hospital Aarhus University Hospital, Aalborg, Denmark, 3 Department of Biotechnology Chemistry and Environmental Engineering Aalborg University, Aalborg, Denmark Introduction: The Affymetrix SNP6.0 genome wide array is commonly used in research for detection of numeric genomic alterations in many haematological malignancies and has been found of prognostic impact. In 2009 Affymetrix launched the cytogenetic 2.7M whole-genome array (Cyto2.7M), which only required 100 ng of genomic DNA (five times less than the SNP6.0 array). Processing time was reduced approximately four times to one day and increased coverage in cancer related genes. The Cyto2.7M array performance on multiple cancer samples has not previously been reported. Materials and Method: We compared the SNP6.0 and the Cyto2.7M types of array on eight cancer samples of diffuse large B-cell lymphoma (DLBCL), known to associate with a large number of deletions and amplifications. Furthermore we evaluated the copy number of selected regions by qPCR. Cyto2.7M arrays were performed on 80 Danish DLBCL’s. Results and Discussion: In the comparison analysis, we identified 226 copy number variations (CNV) of chromosomal gain (42.5%) and loss (57.5%) and of these 199 was present in both array types. To evaluate the CNV boundaries, we calculated the delta break point (DBP) of identified segments, and in 91% of cases the DBP was below 200 kb − the detection limit set in this comparison. The copy number calls of CNVs was analyzed using the smooth signal, and was in all, but 26 events, identical. To determine the actual copy number state in these 26 regions, 6 of these were selected and evaluated by qPCR. Presence of loss of heterozygosity was evaluated for large regions (>10 Mb), and full consistency between arrays was observed. The information obtained from these microarrays can complement data from FISH analysis, as the actual copy number state of a given region is identified; yet the precise translocation is not identified. The occurrence of LOH could suggest the presence of a tumour suppressor gene, although this will have to be validated through further studies. The cytogenetic status of the 80 DLBCL samples were evaluated and compared to published results from Bea et al. 2005. Conclusion: The high degree of consistency between the array types suggests that the Cyto2.7M array is well suited to detect genomic aberrations for cancer samples, and because of the lower input requirements, this could enable genomic analysis of samples where only limited DNA is available. Furthermore, the Cyto2.7M array confirmed the cytogenetic background of DLBCL previously published.
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602 Frequent Epigenetic Inactivation of KEAP1 Gene in Breast Cancer R. Barbano1 , B. Pasculli1 , L.A. Muscarella1 , A. La Torre1 , D. Trombetta1 , R. Murgo2 , V.M. Valori3 , E. Maiello3 , V.M. Fazio4 , P. Parrella4 . 1 IRCCS Casa Sollievo Della Sofferenza, Oncology Laboratory, San Giovanni Rotondo(FG), Italy, 2 IRCCS Casa Sollievo Della Sofferenza, Breast Unit, San Giovanni Rotondo(FG), Italy, 3 IRCCS Casa Sollievo Della Sofferenza, Department of Oncology, San Giovanni Rotondo(FG), Italy, 4 IRCCS Casa Sollievo Della Sofferenza, Laboratory of Oncology, San Giovanni Rotondo(FG), Italy Background: The Nrf2/Keap1 pathway is a master regulator of several redox-sensitive genes implicated in resistance of tumour cells against chemotherapeutic drugs. Recent data suggest that epigenetic mechanisms may play a pivotal role in the regulation of KEAP1 expression. Material and Methods: We determined KEAP1 promoter methylation status in 50 breast cancers (BC), and 6 normal breast tissues (NBT) obtained from reductive mammoplasty. Normal breast tissue adjacent to tumour (NBAT) was available for 45 of the 50 breast cancer cases. In ten cases paired Atypical Ductal Hyperplasia (ADH) and/or Ductal Carcinoma in situ (DCIS) lesions were available along with NBT and tumour tissues. Promoter methylation analysis was performed using a quantitative methylation specific PCR assay in real time (QMSP). Results: Methylation levels were significantly higher in BC (Median 2.3, IQR 0– 17.05) as compared with NBT (Median 0, IQR 0−0) (P = 0.0001 Mann Whitney Test). Overall methylation at the KEAP1 promoter region was detected in 38 out of the 50 BC (76%). Similar level of methylation were detected in normal breast tissues adjacent to tumour (Median 5.27, IQR 0–17.05), ADH (Median 3.85, IQR 1.57–38.46) and DCIS (Median 2.23, IQR 1.27–7.66). Significant different levels of promoter methylation were demonstrated in the three breast cancer subgroups characterized by steroid receptor status and HER2 amplification (triple negative, estrogen receptor positive, and HER2 amplified tumours) (P = 0.04, Kruskall Wallis Test). Conclusions: Our results suggest that deregulation of the Nrf2/Keap1 system could play a pivotal role in the cancerogenesis of breast cancer. Studies are in progress to determine whether KEAP1 abnormalities may contribute to disease progression prediction and response to therapy in these patients. 603 The Effect of p53 on the DNA Repair Enzyme Thymine DNA Glycosylase N. Meireles da Costa1 , A. Hautefeuille2 , M.P. Cros3 , M.E. Melendez4 , P. Swann5 , P. Hainaut2 , L.F. Ribeiro Pinto1 . 1 Instituto Nacional do ˆ ˜ de Medicina Experimental, Rio de Janeiro, Cancer (INCA), Divisao Brazil, 2 International Agency for Research on Cancer (IARC), Molecular Carcinogenesis Group, Lyon, France, 3 International Agency for Research on Cancer (IARC), Epigenetics Group, Lyon, France Background: Thymine DNA glycosylase (TDG) belongs to the superfamily of the uracil DNA glycosylases and is the first enzyme in the base excision repair pathway that removes thymine from the G·T mismatches at CpG sites. Besides its antimutagenic role in DNA repair, the versatile TDG plays a crucial role in maintaining appropriate epigenetic regulation by interacting with transcription factors and histone acetylases, and by acting in the demethylation process of genes previously silenced by promoter methylation and hence reactivating them. Therefore the mechanisms by which proper functioning of TDG is assured may be critical for genetic and epigenetic stability. The tumor supressor TP53 has essential roles in maintaining genomic stability. p53 exerts its control over several DNA-repair pathways, either directly or indirectly. However, so far, there is no information on whether p53 may control TDG. Materials and Methods: Different cell lines mutated in TP53 (MN1 and MDD2 − isogenic breast cancer cell lines harboring either wild-type or inactive p53, respectively; TE-13 and TE-1 − esophageal squamous cell carcinoma cell lines lacking p53 or presenting a temperature-sensitive p53 mutant, respectively) and a normal human bronchial epithelial cell line (NHBE) were used. TP53 was inhibited by using small interfering (si) RNA; increase of p53 levels was induced by transfecting a p53 expression vector; DNA damage was achieved by treating cells with either doxorubicin or MMS. Levels of TDG mRNA and protein expression were analysed after treatments by Quantitative − RT PCR and Western blotting, respectively. Binding of p53 to TDG promoter region was assessed by Chromatin Immunoprecipitation (ChIP) assay and activation of TDG promoter by luciferase assay. Methylation analysis of 6 different gene promoters were performed in samples from 10 Li-Fraumeni TP53 mutation carriers subjects and 10 non-carriers subjects by using pyrosequencing. Results and Discussion: In this study we show that wild-type p53 transcriptionally regulates TDG expression. ChIP and luciferase assays revealed that wild-type p53 binds to a TDG promoter domain containing two p53 consensus response elements (p53RE), inducing its transcription. Using a variety of cell lines we demonstrate that TDG mRNA and protein expression levels are induced by DNA damage in a p53-dependent manner. Although not statistically significant, methylation levels of the analysed gene promoters were found slightly decrease in Li-Fraumeni p53 mutation carriers subjects,