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376: Cancer stem cells, a fuzzy evolving concept: A cell population or a cell property?
S90
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
376 Cancer stem cells, a fuzzy evolving concept: A cell population or a cell property?
379 POLE and POLD1 screening in Portuguese patients with hereditary colorectal cancer
J. Dumont1 , A. Antoniou2 , A. Hebrant2 , C. Maenhaut2 . 1 ERASME, IRIBHM, ´ Brussels, Belgium, 2 Universite´ Libre de Bruxelles Faculte´ de Medecine, IRIBHM, Brussels, Belgium
M. Viegas1 , S. Saraiva2 , M. Areias2 , D. Brito2 , R. Carvalho2 , S. Alves2 , M. Lopes3 , A. Cadime2 , T.C. Martins4 . 1 Portuguese Institute for Oncology at Coimbra (IPO Coimbra FG-EPE), Molecular Pathology Laboratory, Coimbra, Portugal, 2 Portuguese Institute for Oncology at Coimbra (IPO Coimbra FG-EPE), Gastrenterology Service, Coimbra, Portugal, 3 University of Coimbra, Faculty of Pharmacy, Coimbra, Portugal, 4 Portuguese Institute for Oncology at Coimbra (IPO Coimbra FG-EPE), Radiobiology Unit-Medical Physics Service, Coimbra, Portugal
The cancer stem cells (CSC) hypothesis represents a pathological extrapolation of the physiological concept of embryonic and somatic stem cells. In its initial definition, it encompassed the hypothesis of a qualitatively distinct population of immortal cancer cells originating from somatic stem cells, whichgenerate in xenotransplants, by a deterministic irreversible process, the hierarchy of more differentiated finite lifespan derived cells which constitute themselves the bulk of the cancer. These CSC would express specific biomarkers and gene expressions related to chemo and radioresistance, stemness, epithelial mesenchymal transition, etc. No convincing congruence of several of these properties in one cell population has been demonstrated. The concept has greatly evolved with time and with different authors (‘the plasticity of cancer stem cells’) leading to a minimal definition of cells generating a hierarchy of derived cells. In this presentation these concepts are analysed. It is proposed that stemness is a property, more or less reversible, a hallmark of some cells at some time in a cancer cell population, as immortality, dormancy, chemo or radioresistance, epithelial mesenchymal transition etc. These phenotypic properties represent the result of independent, linked or more or less congruent, genetic, epigenetic or signaling programs. No conflict of interest. 378 DNA2 is highly mutated in estrogen-dependent cancers; from a bioinformatics screen to the effect of clinical mutations on cellular growth C. Strauss1 , A. Benvenisty1 , T. Ravid2 , A. Arbel3 , I. Ben-Porath4 , M. Goldberg1 . 1 HUJI, Genetics, Jerusalem, Israel, 2 HUJI, Biochemistry, Jerusalem, Israel, 3 Hadassah Academic College, Medical Laboratory Sciences, Jerusalem, Israel, 4 Hadassah Medical School, Developmental Biology and Cancer Research, Jerusalem, Israel Introduction: A characteristic of cancer is a failure in the DNA damage response (DDR), which contributes to genomic instability and transformation of the cells. All genes found to be involved in familial breast and ovarian cancers participate in different aspects of the DDR. Many DDR genes are regulated by the estrogen hormone. For improving treatment of patients, in this era of personalized cancer therapy, a better understanding of the DDR is required. Also, components of the DDR are potential drug targets for reduced cancer cell viability. Thus, it is important to find novel DDR genes involved in the ethiology of cancer. Materials and Methods: Novel estrogen-dependant cancer genes were found in a computational screen, which scored for DDR genes that are up-regulated by estrogen and highly mutated in breast and ovarian cancers. DNA2, a ‘hit’ of the screen, was further studied by performing genetic, molecular, and computational analyses using yeast, cell cultures and mice as experimental model systems. Results and Discussion: We conducted a screen that presented 5 novel breast and ovarian cancer genes: DNA2, RBL1, RECQL, TIPIN and TOPOR2. DNA2, a helicase involved in DNA replication and DNA end resection repair, was further studied. We found that depletion of DNA2 in tissue culture cells inhibits cellular growth and depletion of DNA2 in cells injected to mice mammary glands results in slower tumor growth and reduced metastasis formation. We also found that high expression of DNA2 correlates with aggressive tumors and shorter life span of patients. Thus, high expression of DNA2, like other helicases, can promote tumorigenesis by facilitate coping with replicative lesions that arise in cancers and with DNA damage caused by radiation or chemotherapy. For understanding how mutations in DNA2 contribute to cancer formation we tested the affect of 4 DNA2 mutations, which were found in ovarian cancer cases, on the activity of DNA2. Using a yeast model, we revealed that these mutations impair human DNA2 activity. DNA2 is upregulated by estrogen, which is known to induce proliferation of cells. We found that, while depletion of DNA2 in cells leads to growth inhibition, supplementing these cells with estrogen restores cell growth. This suggests a model in which cells impaired for DNA2 activity will survive in estrogen expressing tissues. Due to reduction of DNA2 activity, these cells will accumulate mutations, triggering the onset of estrogen-dependant cancers. Conclusion: Here we present 5 novel cancer genes, which we found to be highly mutated in estrogen-dependent cancers and imply that these genes are involved in the etiology of estrogen-dependant cancers. We further characterized one of these genes, DNA2, and demonstrated that it can be implicated in breast and ovarian cancers in both gain and loss of function mechanisms. Our results also suggest that estrogen regulates the cancerous effect of DNA2. No conflict of interest.
Background: Colorectal cancer (CRC) can originate sporadically (around 85%) or as part of hereditary cancer syndromes (approximately 10%). The best studied of these syndromes are Lynch Syndrome (LS; or HNPCC) and Familial Adenomatous Polyposis (FAP). LS families are clinically identified based on the Amsterdam criteria. Genetically, LS is associated with germline mutations in Mismatch Repair (MMR) genes, namely MLH1, MSH2, MSH6 and PMS2, which are associated with defective mismatch repair, leading to microsatellite instability, a hallmark of LS. In turn, FAP is associated with mutations in APC or MutYH. Nevertheless, although some families fulfill the criteria for these syndromes and have a strong probability of carrying mutations in these genes, no pathogenic mutations are found. Moreover, many of these individuals have microsatellite stable tumours. In 2013, two deleterious mutations in POLE (c.1270 c>g) and in POLD1 (c.1433 g>a) were identified and proposed to explain some of these cases of hereditary CRC. POLE and POLD1 encode the catalytic subunits of polymerase e and d, respectively, two enzymes responsible for the synthesis of the leading and the lagging strand of DNA during replication. In this work, we aimed at investigating whether the non-LS, non-FAP, hereditary CRC cases we have in our laboratory were due to mutations in these two genes. Materials and Methods: 76 families were analysed for hereditary CRC from 2009 to 2012. Families that did not present mutations in MMR genes, APC or MutYH, and whose probands were diagnosed with CRC at an early age, had few polyps and microsatellite stable tumours, were selected for screening of POLE and POLD1 mutations. DNA was extracted by phenol/chloroform and ethanol precipitated. The selected probands were screened for POLE and POLD1 described mutations by PCR and automated DNA sequencing. Results: Of the 76 hereditary CRC families, 51.3% had MSI stable tumours and were not tested for mutations in MMR genes. 3.9% presented pathogenic mutations in MMR genes and 14.5% presented MMR variants of unknown significance. No pathogenic mutations were found in APC or MutYH, but variants of unknown clinical significance were found in 3.9% of the cases. Of the 73 cases with no pathogenic mutations, 69 were selected for screening of POLE and POLD1 mutations. None of the individuals screened carried the described POLE or POLD1 mutations. Conclusion: Our preliminary data suggest that POLE and POLD1 mutations are rare in the Portuguese population. No conflict of interest. 380 Characterization of CpG islands and methylation status of Claspin gene (CLSPN) promoter D. Azenha1 , C. Lopes2 , T.C. Martins1 . 1 Portuguese Institute for Oncology at Coimbra (IPO Coimbra FG-EPE), Radiobiology Unit − Medical Physics Service, Coimbra, Portugal, 2 University of Coimbra, Faculty of Pharmacy, Coimbra, Portugal Background: Claspin has a pivotal role in monitoring DNA replication, acting as a sensor that detects active DNA replication complexes and branched DNA structures. In addition, Claspin acts as a mediator in the activation of checkpoint responses and seems to bridge these checkpoints with DNA repair pathways. Therefore, Claspin has an important function in the maintenance of genome stability. Nevertheless, little is known about its role in cancer. We have previously described a series of CLSPN base substitutions that seem to associate with cancer (e.g., breast cancer and gliomas). We have further observed that Claspin expression may be lost in breast tumour cells, suggesting that it may act as a tumour suppressor. As hypermethylation of CpG islands in gene promoters is one of the mechanisms used by cancer cells to silence tumour suppressor genes, we have decided to characterize the CLSPN promoter with regard to CpG islands and to analyse its methylation status in tumour cell lines and paired normal and glioblastoma samples. Material and Methods: The CLSPNpromoter region to analyse was selected based on in silico analysis using different bioinformatic tools. The identification of CpG islands was performed using CpG Plot. DNA was extracted from cell lines (U2OS, HeLa, HEK-293, RKO and U-87 MG) and samples from glioblastoma patients (paired tumour tissue and peripheral blood leukocytes) using phenol-chlorophorm, followed by ethanol precipitation. The methylation status of CpG islands was analysed by bisulphite conversion, using EpiTect Bisulphite Kit, and sequencing, or combined bisulphite restriction analysis (COBRA). Results: We have determined that CLSPN promoter consists of region going from 1 kbp upstream of the initiation codon to 468 bp downstream the initiation
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
376 Cancer stem cells, a fuzzy evolving concept: A cell population or a cell property?
379 POLE and POLD1 screening in Portuguese patients with hereditary colorectal cancer
J. Dumont1 , A. Antoniou2 , A. Hebrant2 , C. Maenhaut2 . 1 ERASME, IRIBHM, ´ Brussels, Belgium, 2 Universite´ Libre de Bruxelles Faculte´ de Medecine, IRIBHM, Brussels, Belgium
M. Viegas1 , S. Saraiva2 , M. Areias2 , D. Brito2 , R. Carvalho2 , S. Alves2 , M. Lopes3 , A. Cadime2 , T.C. Martins4 . 1 Portuguese Institute for Oncology at Coimbra (IPO Coimbra FG-EPE), Molecular Pathology Laboratory, Coimbra, Portugal, 2 Portuguese Institute for Oncology at Coimbra (IPO Coimbra FG-EPE), Gastrenterology Service, Coimbra, Portugal, 3 University of Coimbra, Faculty of Pharmacy, Coimbra, Portugal, 4 Portuguese Institute for Oncology at Coimbra (IPO Coimbra FG-EPE), Radiobiology Unit-Medical Physics Service, Coimbra, Portugal
The cancer stem cells (CSC) hypothesis represents a pathological extrapolation of the physiological concept of embryonic and somatic stem cells. In its initial definition, it encompassed the hypothesis of a qualitatively distinct population of immortal cancer cells originating from somatic stem cells, whichgenerate in xenotransplants, by a deterministic irreversible process, the hierarchy of more differentiated finite lifespan derived cells which constitute themselves the bulk of the cancer. These CSC would express specific biomarkers and gene expressions related to chemo and radioresistance, stemness, epithelial mesenchymal transition, etc. No convincing congruence of several of these properties in one cell population has been demonstrated. The concept has greatly evolved with time and with different authors (‘the plasticity of cancer stem cells’) leading to a minimal definition of cells generating a hierarchy of derived cells. In this presentation these concepts are analysed. It is proposed that stemness is a property, more or less reversible, a hallmark of some cells at some time in a cancer cell population, as immortality, dormancy, chemo or radioresistance, epithelial mesenchymal transition etc. These phenotypic properties represent the result of independent, linked or more or less congruent, genetic, epigenetic or signaling programs. No conflict of interest. 378 DNA2 is highly mutated in estrogen-dependent cancers; from a bioinformatics screen to the effect of clinical mutations on cellular growth C. Strauss1 , A. Benvenisty1 , T. Ravid2 , A. Arbel3 , I. Ben-Porath4 , M. Goldberg1 . 1 HUJI, Genetics, Jerusalem, Israel, 2 HUJI, Biochemistry, Jerusalem, Israel, 3 Hadassah Academic College, Medical Laboratory Sciences, Jerusalem, Israel, 4 Hadassah Medical School, Developmental Biology and Cancer Research, Jerusalem, Israel Introduction: A characteristic of cancer is a failure in the DNA damage response (DDR), which contributes to genomic instability and transformation of the cells. All genes found to be involved in familial breast and ovarian cancers participate in different aspects of the DDR. Many DDR genes are regulated by the estrogen hormone. For improving treatment of patients, in this era of personalized cancer therapy, a better understanding of the DDR is required. Also, components of the DDR are potential drug targets for reduced cancer cell viability. Thus, it is important to find novel DDR genes involved in the ethiology of cancer. Materials and Methods: Novel estrogen-dependant cancer genes were found in a computational screen, which scored for DDR genes that are up-regulated by estrogen and highly mutated in breast and ovarian cancers. DNA2, a ‘hit’ of the screen, was further studied by performing genetic, molecular, and computational analyses using yeast, cell cultures and mice as experimental model systems. Results and Discussion: We conducted a screen that presented 5 novel breast and ovarian cancer genes: DNA2, RBL1, RECQL, TIPIN and TOPOR2. DNA2, a helicase involved in DNA replication and DNA end resection repair, was further studied. We found that depletion of DNA2 in tissue culture cells inhibits cellular growth and depletion of DNA2 in cells injected to mice mammary glands results in slower tumor growth and reduced metastasis formation. We also found that high expression of DNA2 correlates with aggressive tumors and shorter life span of patients. Thus, high expression of DNA2, like other helicases, can promote tumorigenesis by facilitate coping with replicative lesions that arise in cancers and with DNA damage caused by radiation or chemotherapy. For understanding how mutations in DNA2 contribute to cancer formation we tested the affect of 4 DNA2 mutations, which were found in ovarian cancer cases, on the activity of DNA2. Using a yeast model, we revealed that these mutations impair human DNA2 activity. DNA2 is upregulated by estrogen, which is known to induce proliferation of cells. We found that, while depletion of DNA2 in cells leads to growth inhibition, supplementing these cells with estrogen restores cell growth. This suggests a model in which cells impaired for DNA2 activity will survive in estrogen expressing tissues. Due to reduction of DNA2 activity, these cells will accumulate mutations, triggering the onset of estrogen-dependant cancers. Conclusion: Here we present 5 novel cancer genes, which we found to be highly mutated in estrogen-dependent cancers and imply that these genes are involved in the etiology of estrogen-dependant cancers. We further characterized one of these genes, DNA2, and demonstrated that it can be implicated in breast and ovarian cancers in both gain and loss of function mechanisms. Our results also suggest that estrogen regulates the cancerous effect of DNA2. No conflict of interest.
Background: Colorectal cancer (CRC) can originate sporadically (around 85%) or as part of hereditary cancer syndromes (approximately 10%). The best studied of these syndromes are Lynch Syndrome (LS; or HNPCC) and Familial Adenomatous Polyposis (FAP). LS families are clinically identified based on the Amsterdam criteria. Genetically, LS is associated with germline mutations in Mismatch Repair (MMR) genes, namely MLH1, MSH2, MSH6 and PMS2, which are associated with defective mismatch repair, leading to microsatellite instability, a hallmark of LS. In turn, FAP is associated with mutations in APC or MutYH. Nevertheless, although some families fulfill the criteria for these syndromes and have a strong probability of carrying mutations in these genes, no pathogenic mutations are found. Moreover, many of these individuals have microsatellite stable tumours. In 2013, two deleterious mutations in POLE (c.1270 c>g) and in POLD1 (c.1433 g>a) were identified and proposed to explain some of these cases of hereditary CRC. POLE and POLD1 encode the catalytic subunits of polymerase e and d, respectively, two enzymes responsible for the synthesis of the leading and the lagging strand of DNA during replication. In this work, we aimed at investigating whether the non-LS, non-FAP, hereditary CRC cases we have in our laboratory were due to mutations in these two genes. Materials and Methods: 76 families were analysed for hereditary CRC from 2009 to 2012. Families that did not present mutations in MMR genes, APC or MutYH, and whose probands were diagnosed with CRC at an early age, had few polyps and microsatellite stable tumours, were selected for screening of POLE and POLD1 mutations. DNA was extracted by phenol/chloroform and ethanol precipitated. The selected probands were screened for POLE and POLD1 described mutations by PCR and automated DNA sequencing. Results: Of the 76 hereditary CRC families, 51.3% had MSI stable tumours and were not tested for mutations in MMR genes. 3.9% presented pathogenic mutations in MMR genes and 14.5% presented MMR variants of unknown significance. No pathogenic mutations were found in APC or MutYH, but variants of unknown clinical significance were found in 3.9% of the cases. Of the 73 cases with no pathogenic mutations, 69 were selected for screening of POLE and POLD1 mutations. None of the individuals screened carried the described POLE or POLD1 mutations. Conclusion: Our preliminary data suggest that POLE and POLD1 mutations are rare in the Portuguese population. No conflict of interest. 380 Characterization of CpG islands and methylation status of Claspin gene (CLSPN) promoter D. Azenha1 , C. Lopes2 , T.C. Martins1 . 1 Portuguese Institute for Oncology at Coimbra (IPO Coimbra FG-EPE), Radiobiology Unit − Medical Physics Service, Coimbra, Portugal, 2 University of Coimbra, Faculty of Pharmacy, Coimbra, Portugal Background: Claspin has a pivotal role in monitoring DNA replication, acting as a sensor that detects active DNA replication complexes and branched DNA structures. In addition, Claspin acts as a mediator in the activation of checkpoint responses and seems to bridge these checkpoints with DNA repair pathways. Therefore, Claspin has an important function in the maintenance of genome stability. Nevertheless, little is known about its role in cancer. We have previously described a series of CLSPN base substitutions that seem to associate with cancer (e.g., breast cancer and gliomas). We have further observed that Claspin expression may be lost in breast tumour cells, suggesting that it may act as a tumour suppressor. As hypermethylation of CpG islands in gene promoters is one of the mechanisms used by cancer cells to silence tumour suppressor genes, we have decided to characterize the CLSPN promoter with regard to CpG islands and to analyse its methylation status in tumour cell lines and paired normal and glioblastoma samples. Material and Methods: The CLSPNpromoter region to analyse was selected based on in silico analysis using different bioinformatic tools. The identification of CpG islands was performed using CpG Plot. DNA was extracted from cell lines (U2OS, HeLa, HEK-293, RKO and U-87 MG) and samples from glioblastoma patients (paired tumour tissue and peripheral blood leukocytes) using phenol-chlorophorm, followed by ethanol precipitation. The methylation status of CpG islands was analysed by bisulphite conversion, using EpiTect Bisulphite Kit, and sequencing, or combined bisulphite restriction analysis (COBRA). Results: We have determined that CLSPN promoter consists of region going from 1 kbp upstream of the initiation codon to 468 bp downstream the initiation