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513 A Fra-1-regulated Gene Expression Network Required for Maintenance of Epithelial-mesenchymal Transition in Human Colon Cancer Cells
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european journal of cancer 48, suppl. 5 (2012) S25–S288
studied by immunoprecipitation and chromatin structure by nuclease digestion, with HepG2 as a control. Results: Expression of both DLK1 (p = 0.004) and MEG3 (p = 0.01) was significantly diminished in UC tissues and low to undectable in UC cell lines, independent of changes in gene copy numbers. Concomitant loss of expression was accompanied by distinctive methylation patterns at the DMRs and the DLK1 promoter that differed from those of either parental allele. In UC lines, histones at these sites carried modifications indicative of silencing and fixed nucleosomes. In comparison to HepG2 cells, chromatin was poorly accessible to nuclease digestion throughout the region. Conclusions: Decreased expression of DLK1 and MEG3 is highly prevalent in urothelial carcinoma. While allelic losses at 14q32 do occur in UC, the concomitant downregulation of these normally oppositely regulated imprinted genes is probably achieved by epigenetic mechanisms imposing a novel epigenetic state across the region. Supported by the Jurgen-Manchot-Stiftung. ¨ 512 Genetic Aberrations in CDKN2a and TP53 Genes in Patients With Laryngeal Carcinoma T. Todorova1 , S. Jordanov2 , G. Stancheva1 , A. Mitkova1 , I. Chalakov2 , R. Kaneva1 , V. Mitev1 , M. Melnicharov2 , T. Goranova1 . 1 Medical University − Sofia, Molecular Medicine Center, Sofia, Bulgaria, 2 UMHAT “Tsaritsa Yoanna − ISUL”, Department of ENT Clinic of ENT, Sofia, Bulgaria Background: Laryngeal squamous cell carcinoma (LSCC) is the second most common tumour of the head and neck. It is characterized by high rate of developing local or regional recurrence and second primary tumours. Thus, it is of critical importance to understand the molecular basis of laryngeal tumourigenesis. A common feature of laryngeal carcinomas is the inactivation, mainly by mutations, of two tumour-suppressor genes that are involved in cell cycle regulation − CDKN2A and TP53. Methods: In the current study we aimed to examine mutation status of CDKN2A and TP53 genes in a group of 60 patients with laryngeal carcinoma. DNA was extracted from fresh-frozen tumour tissues and exons 1 to 3 of CDKN2A and exons 5 to 8 of TP53 were screened for mutations by direct sequencing. Results: Genetic aberrations in CDKN2A were found in 7 patients (11.7%) and those in TP53 were present in 30 tumours (50%). All but one of the mutations detected in CDKN2A were not described previously. These included two insertions in exon 1 (at positions 34 and 58) and one deletion of 8 bp at position 260_7, one missense (c.343G>T V115L) and two nonsense mutations (c.181G>T E61Stop; c.216 C>A C72Stop) in exon 2. Also, we found 7 novel deletions in TP53 − four in exon 5, one in exon 6 and two in exon 8. No association with clinical features of the patients were found. However, no mutation in any of the two genes was detected in tumours of T stage I and II, implying that they occurred during the cancer progression. Conclusion: Screening for genetic aberrations of patients with carcinoma of the larynx showed a number of novel mutations in CDKN2A and TP53 genes. Our results could contribute to the knowledge about laryngeal carcinogenesis. 513 A Fra-1-regulated Gene Expression Network Required for Maintenance of Epithelial-mesenchymal Transition in Human Colon Cancer Cells J. Diesch1 , E. Sanij1 , J. Ellul1 , I. Haviv2 , C. Love3 , E. Tulchinsky4 , J.M. Mariadason5 , O. Sieber3 , R.D. Hannan1 , A.S. Dhillon1 . 1 Peter MacCallum Cancer Centre, Research Division Oncogenic Signalling and Growth Control Program, Melbourne, Australia, 2 Bar-Ilan University, Faculty of Medicine, Ramat Gan, Israel, 3 Ludwig Institute for Cancer Research, Parkville Branch, Melbourne, Australia, 4 University of Leicester, Cancer Studies and Molecular Medicine, Leicester, United Kingdom, 5 Ludwig Institute for Cancer Research, Austin Health Heidelberg, Melbourne, Australia Background: Colorectal cancer (CRC) is the third most common cancer worldwide with invasion and metastatic spread as primary causes of death from this disease. Fos related antigen-1 (Fra-1) is a component of Activator Protein-1 (AP-1) transcription factor complexes that is frequently over-expressed in a variety of cancers, and our group and others have shown that high Fra-1 levels play a critical role in driving CRC cell migration and invasion. The aims of this study were to unravel genetic programs orchestrated by Fra-1 and identify its direct target genes in invasive CRC cells. Material and Methods: All studies were performed with highly invasive KRAS/BRAF mutant BE CRC cells. Affymetrix Gene 1.0 ST arrays were used for gene expression profiling and chromatin immunoprecipitation (ChIP) followed by high throughput sequencing using Illumina Genome Analyzer II to identify genomic loci occupied by Fra-1. Results: Stable silencing of Fra-1 expression in BE CRC cells resulted in dramatic morphological changes, with the cells switching from a mesenchymal
Sunday 8 − Tuesday 10 July 2012
phenotype to an epithelial-like appearance and complete inhibition of cell migration and invasion. To determine how Fra-1 silencing invokes this striking phenotypic switch, we searched for its transcriptional targets in BE cells using a combination of expression arrays to identify Fra-1-regulated genes, and ChIP-Seq (ChIP assay coupled with high throughput sequencing) to identify genomic binding sites of Fra-1. We found that Fra-1 is bound in close proximity to the transcription start site of many genes identified in the expression arrays and that the largest ontological class of genes overrepresented in both datasets encoded proteins involved in a process describes as epithelial-mesenchymal transition (EMT) (herein termed Fra-1EMT genes). Amongst these, genes associated with a mesenchymal expression profile were suppressed upon Fra-1 silencing, whereas genes fitting an epithelial profile were up-regulated. Thus, stable silencing of Fra-1 leads to a reversal of EMT in BE cells. To determine whether changes in the expression of Fra-1EMT genes occur in primary CRCs, we performed unsupervised clustering on existing microarray data from 185 stage B and C CRCs. This analysis demonstrated that the Fra-1EMT signature is associated with poor prognosis in CRC patients. Interestingly, histochemical staining of Fra-1 in CRC specimens revealed that Fra-1 expression is significantly enriched in budding tumour cells. Tumour buds represent the invasive front of colorectal tumours and more importantly are thought to be the histomorphological hallmarks of EMT in CRCs. Conclusions: In this study we demonstrate that Fra-1 is a key regulator of transcriptional programs required to maintain EMT in CRC cells. Furthermore, this is the first report that shows a direct link between the pattern of Fra-1 expression and EMT at the invasive front of CRC, and thus establishes Fra-1 as a new histochemical marker for a subset of highly aggressive CRCs. 514 Alterations of Genome Methylation Impact Tumoral Progression in Human Prolactinoma M. Roche1 , N. Nazaret2 , S. Croze2 , F. Barbet2 , C. Rey2 , C. Legras-Lachuer3 , J. Trouillas4 , A. Wierinckx1 , J. Lachuer1 . 1 Cancerology Research Center of Lyon (umr inserm 1052 cnrs 5286 ucbl1), Tumoral Escape, Lyon, France, 2 Universite´ Lyon 1 sfr est, ProfileXpert, Lyon, France, 3 Umr Cnrs 5557 Ucbl Usc Inra 1193 Envl, Dynamique Microbienne et Transmission Virale, Lyon, France, 4 Centre de Neurosciences de Lyon, Neuro-oncologie Neuro-inflammation, Lyon, France Alterations of DNA methylation pattern, and especially methylation of promoter, are described as one of the key mechanism involved in tumoral initiation. Nevertheless, the role of these changes in tumoral progression remains unclear. To provide new clues about the role of DNA methylation alterations in tumoral progression, we carried out the impact of promoter methylation on gene expression in non-aggressive (NA) and aggressive including malignant (A) human prolactinomas. To investigate this fundamental question, we used a multi-dimensional integrative strategy combining transcriptomic and epigenetics data obtained on the same human tumors using genome wide approaches. Global DNA methylation profiling was realized using whole genome bisulfite-sequencing by NGS technology (HiSeq 2000, Illumina® ) on one A and one NA tumor. Transcriptomic activity was assessed using Codelink® Human Whole Genome microarray on the same tumors and was confirmed on an extensive cohort. Methylation levels of distal promoters (10kb to 1 kb from TSS) and TSS regions (1kb to +1kb from TSS) were compared between the two phenotypes of tumors. It revealed that 1016 distal promoters and 800 TSS regions were differentially methylated between A and NA tumors. Moreover, the majority of the differentially methylated regions were hypermethylated in A vs. NA tumor. Integrative analysis between transcriptomic activity and methylation patterns showed that about 20% of differentially methylated TSS regions and 16% of differentially methylated distal promoters were inversely correlated to gene expression variation. Interestingly this analysis also highlighted that hypermethylation of TSS region but not distal promoter was linked to enrichment in down-regulated genes. Expression of genes regulated by differential DNA methylation between the two tumors was investigated on an extensive cohort leading to the selection of genes with similar expression levels. Here we performed the first integrative analysis including whole genome bisulfite sequencing and transcriptomic activity on the same human PRL tumors. Interestingly, our results suggested that hypermethylation of TSS region is more efficient for gene repression than distal promoter methylation. Moreover, this study allowed the fine selection of specific genes on the basis of their promoters’ methylated pattern. It appeared that these genes are mainly involved in proliferation control and linked to malignant progression and would be precisely analyzed on more prolactinomas to confirm the involvement of DNA methylation in their deregulation.
european journal of cancer 48, suppl. 5 (2012) S25–S288
studied by immunoprecipitation and chromatin structure by nuclease digestion, with HepG2 as a control. Results: Expression of both DLK1 (p = 0.004) and MEG3 (p = 0.01) was significantly diminished in UC tissues and low to undectable in UC cell lines, independent of changes in gene copy numbers. Concomitant loss of expression was accompanied by distinctive methylation patterns at the DMRs and the DLK1 promoter that differed from those of either parental allele. In UC lines, histones at these sites carried modifications indicative of silencing and fixed nucleosomes. In comparison to HepG2 cells, chromatin was poorly accessible to nuclease digestion throughout the region. Conclusions: Decreased expression of DLK1 and MEG3 is highly prevalent in urothelial carcinoma. While allelic losses at 14q32 do occur in UC, the concomitant downregulation of these normally oppositely regulated imprinted genes is probably achieved by epigenetic mechanisms imposing a novel epigenetic state across the region. Supported by the Jurgen-Manchot-Stiftung. ¨ 512 Genetic Aberrations in CDKN2a and TP53 Genes in Patients With Laryngeal Carcinoma T. Todorova1 , S. Jordanov2 , G. Stancheva1 , A. Mitkova1 , I. Chalakov2 , R. Kaneva1 , V. Mitev1 , M. Melnicharov2 , T. Goranova1 . 1 Medical University − Sofia, Molecular Medicine Center, Sofia, Bulgaria, 2 UMHAT “Tsaritsa Yoanna − ISUL”, Department of ENT Clinic of ENT, Sofia, Bulgaria Background: Laryngeal squamous cell carcinoma (LSCC) is the second most common tumour of the head and neck. It is characterized by high rate of developing local or regional recurrence and second primary tumours. Thus, it is of critical importance to understand the molecular basis of laryngeal tumourigenesis. A common feature of laryngeal carcinomas is the inactivation, mainly by mutations, of two tumour-suppressor genes that are involved in cell cycle regulation − CDKN2A and TP53. Methods: In the current study we aimed to examine mutation status of CDKN2A and TP53 genes in a group of 60 patients with laryngeal carcinoma. DNA was extracted from fresh-frozen tumour tissues and exons 1 to 3 of CDKN2A and exons 5 to 8 of TP53 were screened for mutations by direct sequencing. Results: Genetic aberrations in CDKN2A were found in 7 patients (11.7%) and those in TP53 were present in 30 tumours (50%). All but one of the mutations detected in CDKN2A were not described previously. These included two insertions in exon 1 (at positions 34 and 58) and one deletion of 8 bp at position 260_7, one missense (c.343G>T V115L) and two nonsense mutations (c.181G>T E61Stop; c.216 C>A C72Stop) in exon 2. Also, we found 7 novel deletions in TP53 − four in exon 5, one in exon 6 and two in exon 8. No association with clinical features of the patients were found. However, no mutation in any of the two genes was detected in tumours of T stage I and II, implying that they occurred during the cancer progression. Conclusion: Screening for genetic aberrations of patients with carcinoma of the larynx showed a number of novel mutations in CDKN2A and TP53 genes. Our results could contribute to the knowledge about laryngeal carcinogenesis. 513 A Fra-1-regulated Gene Expression Network Required for Maintenance of Epithelial-mesenchymal Transition in Human Colon Cancer Cells J. Diesch1 , E. Sanij1 , J. Ellul1 , I. Haviv2 , C. Love3 , E. Tulchinsky4 , J.M. Mariadason5 , O. Sieber3 , R.D. Hannan1 , A.S. Dhillon1 . 1 Peter MacCallum Cancer Centre, Research Division Oncogenic Signalling and Growth Control Program, Melbourne, Australia, 2 Bar-Ilan University, Faculty of Medicine, Ramat Gan, Israel, 3 Ludwig Institute for Cancer Research, Parkville Branch, Melbourne, Australia, 4 University of Leicester, Cancer Studies and Molecular Medicine, Leicester, United Kingdom, 5 Ludwig Institute for Cancer Research, Austin Health Heidelberg, Melbourne, Australia Background: Colorectal cancer (CRC) is the third most common cancer worldwide with invasion and metastatic spread as primary causes of death from this disease. Fos related antigen-1 (Fra-1) is a component of Activator Protein-1 (AP-1) transcription factor complexes that is frequently over-expressed in a variety of cancers, and our group and others have shown that high Fra-1 levels play a critical role in driving CRC cell migration and invasion. The aims of this study were to unravel genetic programs orchestrated by Fra-1 and identify its direct target genes in invasive CRC cells. Material and Methods: All studies were performed with highly invasive KRAS/BRAF mutant BE CRC cells. Affymetrix Gene 1.0 ST arrays were used for gene expression profiling and chromatin immunoprecipitation (ChIP) followed by high throughput sequencing using Illumina Genome Analyzer II to identify genomic loci occupied by Fra-1. Results: Stable silencing of Fra-1 expression in BE CRC cells resulted in dramatic morphological changes, with the cells switching from a mesenchymal
Sunday 8 − Tuesday 10 July 2012
phenotype to an epithelial-like appearance and complete inhibition of cell migration and invasion. To determine how Fra-1 silencing invokes this striking phenotypic switch, we searched for its transcriptional targets in BE cells using a combination of expression arrays to identify Fra-1-regulated genes, and ChIP-Seq (ChIP assay coupled with high throughput sequencing) to identify genomic binding sites of Fra-1. We found that Fra-1 is bound in close proximity to the transcription start site of many genes identified in the expression arrays and that the largest ontological class of genes overrepresented in both datasets encoded proteins involved in a process describes as epithelial-mesenchymal transition (EMT) (herein termed Fra-1EMT genes). Amongst these, genes associated with a mesenchymal expression profile were suppressed upon Fra-1 silencing, whereas genes fitting an epithelial profile were up-regulated. Thus, stable silencing of Fra-1 leads to a reversal of EMT in BE cells. To determine whether changes in the expression of Fra-1EMT genes occur in primary CRCs, we performed unsupervised clustering on existing microarray data from 185 stage B and C CRCs. This analysis demonstrated that the Fra-1EMT signature is associated with poor prognosis in CRC patients. Interestingly, histochemical staining of Fra-1 in CRC specimens revealed that Fra-1 expression is significantly enriched in budding tumour cells. Tumour buds represent the invasive front of colorectal tumours and more importantly are thought to be the histomorphological hallmarks of EMT in CRCs. Conclusions: In this study we demonstrate that Fra-1 is a key regulator of transcriptional programs required to maintain EMT in CRC cells. Furthermore, this is the first report that shows a direct link between the pattern of Fra-1 expression and EMT at the invasive front of CRC, and thus establishes Fra-1 as a new histochemical marker for a subset of highly aggressive CRCs. 514 Alterations of Genome Methylation Impact Tumoral Progression in Human Prolactinoma M. Roche1 , N. Nazaret2 , S. Croze2 , F. Barbet2 , C. Rey2 , C. Legras-Lachuer3 , J. Trouillas4 , A. Wierinckx1 , J. Lachuer1 . 1 Cancerology Research Center of Lyon (umr inserm 1052 cnrs 5286 ucbl1), Tumoral Escape, Lyon, France, 2 Universite´ Lyon 1 sfr est, ProfileXpert, Lyon, France, 3 Umr Cnrs 5557 Ucbl Usc Inra 1193 Envl, Dynamique Microbienne et Transmission Virale, Lyon, France, 4 Centre de Neurosciences de Lyon, Neuro-oncologie Neuro-inflammation, Lyon, France Alterations of DNA methylation pattern, and especially methylation of promoter, are described as one of the key mechanism involved in tumoral initiation. Nevertheless, the role of these changes in tumoral progression remains unclear. To provide new clues about the role of DNA methylation alterations in tumoral progression, we carried out the impact of promoter methylation on gene expression in non-aggressive (NA) and aggressive including malignant (A) human prolactinomas. To investigate this fundamental question, we used a multi-dimensional integrative strategy combining transcriptomic and epigenetics data obtained on the same human tumors using genome wide approaches. Global DNA methylation profiling was realized using whole genome bisulfite-sequencing by NGS technology (HiSeq 2000, Illumina® ) on one A and one NA tumor. Transcriptomic activity was assessed using Codelink® Human Whole Genome microarray on the same tumors and was confirmed on an extensive cohort. Methylation levels of distal promoters (10kb to 1 kb from TSS) and TSS regions (1kb to +1kb from TSS) were compared between the two phenotypes of tumors. It revealed that 1016 distal promoters and 800 TSS regions were differentially methylated between A and NA tumors. Moreover, the majority of the differentially methylated regions were hypermethylated in A vs. NA tumor. Integrative analysis between transcriptomic activity and methylation patterns showed that about 20% of differentially methylated TSS regions and 16% of differentially methylated distal promoters were inversely correlated to gene expression variation. Interestingly this analysis also highlighted that hypermethylation of TSS region but not distal promoter was linked to enrichment in down-regulated genes. Expression of genes regulated by differential DNA methylation between the two tumors was investigated on an extensive cohort leading to the selection of genes with similar expression levels. Here we performed the first integrative analysis including whole genome bisulfite sequencing and transcriptomic activity on the same human PRL tumors. Interestingly, our results suggested that hypermethylation of TSS region is more efficient for gene repression than distal promoter methylation. Moreover, this study allowed the fine selection of specific genes on the basis of their promoters’ methylated pattern. It appeared that these genes are mainly involved in proliferation control and linked to malignant progression and would be precisely analyzed on more prolactinomas to confirm the involvement of DNA methylation in their deregulation.