- Email: [email protected]
580 Identification of Functional Targets of Serous Ovarian Cancer
S138
european journal of cancer 48, suppl. 5 (2012) S25–S288
epigenetic mechanisms can provide attractive targets for prenatal modulation and prevention of inflammatory and other related human diseases. 578 Detection of Hot Spot Mutations by Semi-conductor Sequencing of DNA Extracted From Formalin Fixed Paraffin Embedded (FFPE) Colon Rectal Cancer Samples A. Rico1 , A. Didelot2 , P. Rosella3 , H. Blons2 , P. Laurent-Puig2 . 1 Life Technologies, Falcon Team, Saint Aubin, France, 2 Universite´ Paris Descartes, UMR-S775, Paris, France, 3 Life Technologies, Falcon Team, Paris, France The characterization of gene mutations in solid tumor samples is now a part of the clinical management of cancer patients in order to determine the best therapeutic option and it opens new avenue in the personalized medicine approach of cancer treatment. Up to now, different molecular biological techniques have been developed in order to try to answer to the increase number of tests needed to be able to take a therapeutic decision and to deal with limitations: the poor quality of the DNA extracted most of the time from formalin fixed paraffin embedded (FFPE) tissues, the low quantity of DNA available, and the contamination of tumor sample by non-tumor cells. Alternative methods to the sanger sequencing are sensitive but limited in terms of number of mutation hotspots explored as well as the quantity of DNA necessary to do the different tests. The next generation sequencing systems overcome most of these limitation by allowing the exploration of a large portion of targeted genes, with a low DNA quantity used and the high number of possible reads increasing the sensitivity of the method. We tested 8 FFPE tumors samples derived from 6 lung cancers and 2 colon cancers. These tumors have been previously characterized for KRAS and EGFR mutation by TaqMan® Mutation Detection Assays based on Competitive Allele-Specific PCR technology (castPCR, LifeTechnologies). Five tumors were mutated for KRAS (2 colon and 3 lung cancers) and 3 were mutated for EGFR (3 lung cancers). The DNA was extracted from FFPE samples using Qiagen kit. The AmpliSeq™ Cancer Panel from Life Technologies was used allowing the amplification of different targeted region including those of KRAS and EGFR. A maximum of 10 nanograms of DNA were used as template for each tumor. Four tumors were sequenced using the Ion AmpliSeq™ Cancer Panel and Ion 314 chip on the Ion Torrent™ PGM™ (1 tumor per chip) and 4 tumors were sequenced using a unique Ion 316 chip. In this last case we used barcodes to distinguish the different sample from each other. The sequences were analyzed using the Variant Caller plug-in of the PGM™ Torrent Server and a report was automatically generated including links to IGV software tool to view the sequences. The mean number of mapped reads per tumor was 399,005±44238, the average base coverage depth per tumor was 1809±357, the coverage at 100x was observed in 86.7±4.4% of the targeted sequences. All the predefined mutations in KRAS and EGFR were found. Additional mutations were found in APC, TP53, CTNNB1 and PIK3CA genes. Furthermore known polymorphisms were characterized in different genes. The coverage of targeted sequence appear to be enough to provide a sensitivity of 5%. The Ion AmpliSeq™ Cancer Panel tested seems to provide a cost effectiveness alternative to characterize lung and colon tumor samples in translational cancer research laboratory. 579 Parthenolide Inhibits Tumor Promotion − Epigenetic Regulation of P21 N. Darwiche1 , A. Ghantous2 , M. Saikali1 , T. Rau3 , R. Schneider-Stock3 . 1 American University of Beirut, Biochemistry and Molecular Genetics Dept., Beirut, Lebanon, 2 American University of Beirut, Biology Dept., Beirut, Lebanon, 3 University Erlangen-Nuremberg, Institute for Pathology, Erlangen, Germany Background: The promotion stage in the multistep process of tumorigenesis is NF-kB-dependent, epigenetically regulated, and reversible, thus, a suitable target for chemoprevention. We investigated whether the NF-kB inhibitor, parthenolide, currently in cancer clinical trials, attenuates tumor promotion by modulating the epigenetically regulated NF-kB target genes, p21 and cyclin D1, which play crucial roles in epidermal tumor promotion and carcinogenesis. Material and Methods: Cell growth assays, soft agar colony formation, cell cycle analyses by flow cytometry, NF-kB gel shift and luciferase reporter assays, westerns, real-time PCR, chromatin immuno-precipitation (ChIP) assays, siRNA, tumor mouse models, tissue microarrays. Results: Parthenolide selectively inhibited the growth of neoplastic keratinocytes while sparing normal cells using in vitro models of human and murine epidermal carcinogenesis. In JB6P+ cells, a model of tumor promotion, parthenolide abrogated promoter-induced cell proliferation and anchorageindependent growth, blocking promoted cells in S-G2/M phases. Furthermore, parthenolide decreased basal and promoter-induced NF-kB activity and modulated the expression of the NF-kB target genes, p21 and cyclin D1. In parthenolide-treated cells, p21 transcription correlated more with relaxed chromatin than with p65/NF-kB binding at the p21 promoter. However, cyclin D1 transcription correlated more with p65/NF-kB binding than with chromatin structure at the cyclin D1 promoter. Using p21-siRNA and human colon
Sunday 8 − Tuesday 10 July 2012
carcinoma HCT-116 wild type and p21−/− clonal cell variants, we showed that p21 mediates cell sensitivity to parthenolide. Finally, parthenolide did not alter general histone deacetylase (HDAC) activity or global chromatin structure and synergistically inhibited the growth of epidermal tumor cells when combined with HDAC but not DNA methyltransferase inhibitors that are used in cancer clinical trials. In vivo, low parthenolide concentrations (0.25 mg/Kg) inhibited tumor growth of promoted JB6P+ cells in xenograft NMRI immuno-compromised mice, when administered intraperitoneally every other day over a ten-day period and stopped upon injection of tumor cells or stopped upon tumor appearance. Tissue microarray of mouse tumors showed that parthenolide decreased tumor cell proliferation, evaluated by Ki67, decreased p65/NF-kB levels, and increased p21 expression. Conclusion: These results show that, at low doses, parthenolide inhibits tumor promotion and epigenetically regulates the expression of the NF-kB target gene, p21, which is crucial for parthenolide’s antitumor potential. Parthenolide is currently in cancer clinical trials and our result support its testing in epigenetic therapy whether alone or in combination with other drugs. 580 Identification of Functional Targets of Serous Ovarian Cancer Q.H. Miow1 , T.Z. Tan2 , J. Ye2 , J.A. Lau2 , J.P. Thiery2 , S. Mori2 . 1 National University of Singapore, NUS Graduate School for Integrative Sciences and Engineering, Singapore, Singapore, 2 National University of Singapore, Cancer Science Institute of Singapore, Singapore The development of more effective regimens is critical to achieve major advances in ovarian cancer therapeutics. In order to address the extensive heterogeneity exhibited within the disease of ovarian cancer, we first developed an expression-based classification scheme to further subdivide high-grade ovarian serous adenocarcinoma into five distinct subtypes (Epi-A, Epi-B, Mes, Stem-A and Stem-B) exhibiting more biological and clinical homogeneity. This classification scheme now provides us an opportunity to identify molecular targets specific for each of the multiple subtypes. Based on this scheme, we performed genome-wide functional screens to identify subtype-specific growthpromoting genes through the use of pooled shRNA lentivirus library. 88, 85 and 77 genes were found to be potentially important for the cell growth of Stem-A, Mes and Epi-A cell lines, respectively. Upon validation with individual siRNA, five genes were identified to be specifically relevant for cell growth in Stem-A subtype, which exhibits poorer patient outcomes in clinics. Although additional studies are still required to provide insights on the mechanism, our findings demonstrated that there are indeed distinct patterns of growth determinants across the transcriptional subtypes, therefore implied a potential to develop subtype-specific regimens in the therapeutic of serous ovarian cancer. 581 CpG Methylation and Transcription Factor C-Myb Regulate Human Vav1 Expression in Hematopoietic and Cancer Cell Lines L. Ilan1 , S. Katzav1 . 1 Hebrew University Medical School, Developmental Biology & Cancer Research IMRIC, Jerusalem, Israel Introduction: Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA). Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression in non-hematological tissues is completely understood. In the present study, we focused on understanding the mechanisms regulating vav1 gene transcription in hematopoietic and lung cancer cell lines. Material and Methods: site-directed mutagenesis, luciferase reporter assay, bisulfite sequencing, electrophoretic mobility shift assay and western blotting were used in our studies. Results and Discussion: We demonstrate that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but shows various degree of methylation in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a single CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Conclusion: Our data reveal two regulatory mechanisms for vav1 expression: binding of c-Myb and interruption of transcription factors binding by CpG
european journal of cancer 48, suppl. 5 (2012) S25–S288
epigenetic mechanisms can provide attractive targets for prenatal modulation and prevention of inflammatory and other related human diseases. 578 Detection of Hot Spot Mutations by Semi-conductor Sequencing of DNA Extracted From Formalin Fixed Paraffin Embedded (FFPE) Colon Rectal Cancer Samples A. Rico1 , A. Didelot2 , P. Rosella3 , H. Blons2 , P. Laurent-Puig2 . 1 Life Technologies, Falcon Team, Saint Aubin, France, 2 Universite´ Paris Descartes, UMR-S775, Paris, France, 3 Life Technologies, Falcon Team, Paris, France The characterization of gene mutations in solid tumor samples is now a part of the clinical management of cancer patients in order to determine the best therapeutic option and it opens new avenue in the personalized medicine approach of cancer treatment. Up to now, different molecular biological techniques have been developed in order to try to answer to the increase number of tests needed to be able to take a therapeutic decision and to deal with limitations: the poor quality of the DNA extracted most of the time from formalin fixed paraffin embedded (FFPE) tissues, the low quantity of DNA available, and the contamination of tumor sample by non-tumor cells. Alternative methods to the sanger sequencing are sensitive but limited in terms of number of mutation hotspots explored as well as the quantity of DNA necessary to do the different tests. The next generation sequencing systems overcome most of these limitation by allowing the exploration of a large portion of targeted genes, with a low DNA quantity used and the high number of possible reads increasing the sensitivity of the method. We tested 8 FFPE tumors samples derived from 6 lung cancers and 2 colon cancers. These tumors have been previously characterized for KRAS and EGFR mutation by TaqMan® Mutation Detection Assays based on Competitive Allele-Specific PCR technology (castPCR, LifeTechnologies). Five tumors were mutated for KRAS (2 colon and 3 lung cancers) and 3 were mutated for EGFR (3 lung cancers). The DNA was extracted from FFPE samples using Qiagen kit. The AmpliSeq™ Cancer Panel from Life Technologies was used allowing the amplification of different targeted region including those of KRAS and EGFR. A maximum of 10 nanograms of DNA were used as template for each tumor. Four tumors were sequenced using the Ion AmpliSeq™ Cancer Panel and Ion 314 chip on the Ion Torrent™ PGM™ (1 tumor per chip) and 4 tumors were sequenced using a unique Ion 316 chip. In this last case we used barcodes to distinguish the different sample from each other. The sequences were analyzed using the Variant Caller plug-in of the PGM™ Torrent Server and a report was automatically generated including links to IGV software tool to view the sequences. The mean number of mapped reads per tumor was 399,005±44238, the average base coverage depth per tumor was 1809±357, the coverage at 100x was observed in 86.7±4.4% of the targeted sequences. All the predefined mutations in KRAS and EGFR were found. Additional mutations were found in APC, TP53, CTNNB1 and PIK3CA genes. Furthermore known polymorphisms were characterized in different genes. The coverage of targeted sequence appear to be enough to provide a sensitivity of 5%. The Ion AmpliSeq™ Cancer Panel tested seems to provide a cost effectiveness alternative to characterize lung and colon tumor samples in translational cancer research laboratory. 579 Parthenolide Inhibits Tumor Promotion − Epigenetic Regulation of P21 N. Darwiche1 , A. Ghantous2 , M. Saikali1 , T. Rau3 , R. Schneider-Stock3 . 1 American University of Beirut, Biochemistry and Molecular Genetics Dept., Beirut, Lebanon, 2 American University of Beirut, Biology Dept., Beirut, Lebanon, 3 University Erlangen-Nuremberg, Institute for Pathology, Erlangen, Germany Background: The promotion stage in the multistep process of tumorigenesis is NF-kB-dependent, epigenetically regulated, and reversible, thus, a suitable target for chemoprevention. We investigated whether the NF-kB inhibitor, parthenolide, currently in cancer clinical trials, attenuates tumor promotion by modulating the epigenetically regulated NF-kB target genes, p21 and cyclin D1, which play crucial roles in epidermal tumor promotion and carcinogenesis. Material and Methods: Cell growth assays, soft agar colony formation, cell cycle analyses by flow cytometry, NF-kB gel shift and luciferase reporter assays, westerns, real-time PCR, chromatin immuno-precipitation (ChIP) assays, siRNA, tumor mouse models, tissue microarrays. Results: Parthenolide selectively inhibited the growth of neoplastic keratinocytes while sparing normal cells using in vitro models of human and murine epidermal carcinogenesis. In JB6P+ cells, a model of tumor promotion, parthenolide abrogated promoter-induced cell proliferation and anchorageindependent growth, blocking promoted cells in S-G2/M phases. Furthermore, parthenolide decreased basal and promoter-induced NF-kB activity and modulated the expression of the NF-kB target genes, p21 and cyclin D1. In parthenolide-treated cells, p21 transcription correlated more with relaxed chromatin than with p65/NF-kB binding at the p21 promoter. However, cyclin D1 transcription correlated more with p65/NF-kB binding than with chromatin structure at the cyclin D1 promoter. Using p21-siRNA and human colon
Sunday 8 − Tuesday 10 July 2012
carcinoma HCT-116 wild type and p21−/− clonal cell variants, we showed that p21 mediates cell sensitivity to parthenolide. Finally, parthenolide did not alter general histone deacetylase (HDAC) activity or global chromatin structure and synergistically inhibited the growth of epidermal tumor cells when combined with HDAC but not DNA methyltransferase inhibitors that are used in cancer clinical trials. In vivo, low parthenolide concentrations (0.25 mg/Kg) inhibited tumor growth of promoted JB6P+ cells in xenograft NMRI immuno-compromised mice, when administered intraperitoneally every other day over a ten-day period and stopped upon injection of tumor cells or stopped upon tumor appearance. Tissue microarray of mouse tumors showed that parthenolide decreased tumor cell proliferation, evaluated by Ki67, decreased p65/NF-kB levels, and increased p21 expression. Conclusion: These results show that, at low doses, parthenolide inhibits tumor promotion and epigenetically regulates the expression of the NF-kB target gene, p21, which is crucial for parthenolide’s antitumor potential. Parthenolide is currently in cancer clinical trials and our result support its testing in epigenetic therapy whether alone or in combination with other drugs. 580 Identification of Functional Targets of Serous Ovarian Cancer Q.H. Miow1 , T.Z. Tan2 , J. Ye2 , J.A. Lau2 , J.P. Thiery2 , S. Mori2 . 1 National University of Singapore, NUS Graduate School for Integrative Sciences and Engineering, Singapore, Singapore, 2 National University of Singapore, Cancer Science Institute of Singapore, Singapore The development of more effective regimens is critical to achieve major advances in ovarian cancer therapeutics. In order to address the extensive heterogeneity exhibited within the disease of ovarian cancer, we first developed an expression-based classification scheme to further subdivide high-grade ovarian serous adenocarcinoma into five distinct subtypes (Epi-A, Epi-B, Mes, Stem-A and Stem-B) exhibiting more biological and clinical homogeneity. This classification scheme now provides us an opportunity to identify molecular targets specific for each of the multiple subtypes. Based on this scheme, we performed genome-wide functional screens to identify subtype-specific growthpromoting genes through the use of pooled shRNA lentivirus library. 88, 85 and 77 genes were found to be potentially important for the cell growth of Stem-A, Mes and Epi-A cell lines, respectively. Upon validation with individual siRNA, five genes were identified to be specifically relevant for cell growth in Stem-A subtype, which exhibits poorer patient outcomes in clinics. Although additional studies are still required to provide insights on the mechanism, our findings demonstrated that there are indeed distinct patterns of growth determinants across the transcriptional subtypes, therefore implied a potential to develop subtype-specific regimens in the therapeutic of serous ovarian cancer. 581 CpG Methylation and Transcription Factor C-Myb Regulate Human Vav1 Expression in Hematopoietic and Cancer Cell Lines L. Ilan1 , S. Katzav1 . 1 Hebrew University Medical School, Developmental Biology & Cancer Research IMRIC, Jerusalem, Israel Introduction: Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA). Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression in non-hematological tissues is completely understood. In the present study, we focused on understanding the mechanisms regulating vav1 gene transcription in hematopoietic and lung cancer cell lines. Material and Methods: site-directed mutagenesis, luciferase reporter assay, bisulfite sequencing, electrophoretic mobility shift assay and western blotting were used in our studies. Results and Discussion: We demonstrate that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but shows various degree of methylation in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a single CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Conclusion: Our data reveal two regulatory mechanisms for vav1 expression: binding of c-Myb and interruption of transcription factors binding by CpG