- Email: [email protected]
567: The impact of chronic cisplatin treatment on DNA repair in head and neck squamous cell carcinoma
S136
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
566 Investigating PI3Kinase and K-ras pathway interactions in mouse model of colorectal cancer V. Meniel1 , I. Martin-Berenjeno2 , B. Vanhaesebroeck2 , A.R. Clarke1 . 1 Cardiff University, European Cancer Stem Cell Research Institute, Cardiff Wales, United Kingdom, 2 University College London, UCL Cancer Institute, London, United Kingdom Introduction: K-Ras, the p110a catalytic subunit of PI3K (PIK3CA), and the negative regulator, PTEN, are all frequently altered in colorectal cancer. Mutation in these genes leads to aberrant activation of PI3K/Akt and MAPK/Erk signaling pathways. 21.3% of colorectal cancers with K-ras or B-raf mutations have concomitant mutations with PI3KCA whereas PTEN loss and PI3KCA mutations have been shown to co-exist in 6.6% colorectal cancers. Previously we have shown that PTEN loss in the mouse leads to increased tumorigenicity in the context of Apc deficiency and similarly activating mutation K-rasV12 accelerated intestinal tumorigenesis and progression. Material and Methods: Using our Cre-lox Apc mouse model, we are now investigating the effect on intestinal tumorigenesis and progression after dual Wnt signalling activation and PI3KCA oncogenic activation using the H1047R hot spot activating mutation (P110aonc). To study the synergy between aberrant activation of PI3K/Akt pathway and MAPK/Erk patwhay, we are also studying the additional effect of K-ras activated mutation or Pten deficiency in the context of PI3KCA activation together with loss of Apc. Results and Discussion: We are currently generating cohorts of mice Flp-P110oncVillinApcfl/+ , Flp-P110aoncVillinApcfl/+ Ptenfl/fl , Flp-P110aoncVillinApcfl/+ KrasV12 together with appropriate controls. Also, we are studying the role of oncogenic activation of PI3KCA in intestinal homeostasis. Preliminary data indicates that tamoxifene induced P110aonc Apcfl/+ mice have a shorter survival compared to tamoxifene induced Apcfl/+ mice. The histopathology, tumour burden and tumour progression analysis of all cohorts are in the process of investigation. No conflict of interest. 567 The impact of chronic cisplatin treatment on DNA repair in head and neck squamous cell carcinoma W. Su1 , W.C. Su1 , J.Y. Chang1 , H.J. Liaw2 . 1 National Cheng Kong University Hospital, Department of Internal Medicine, Tainan City, Taiwan, 2 National Cheng Kong University Hospital, Department of Life Science, Tainan City, Taiwan Introduction: Cisplatin is an important chemotherapeutic agent for many cancers, including head and neck squamous cell carcinoma (HNSCC). Cisplatin cross-links DNA and then interferes with cell division by mitosis. Repeated cisplatin treatment is a way to treat recurrent HNSCC; however, the long-term effect of chronic cisplatin treatment is unknown. We aimed to determine whether chronic cisplatin treatment induced more ability of DNA repair. Material and Methods: Human nasopharyngeal carcinoma cells (HONE-1) and its cisplatin resistant HONE-6 cells were gifts from Prof. Chang JangYang. HONE-6 cells were developed through repeated selection from the parental HONE-1 cells using cisplatin containing medium. We treated HONE-1 and HONE-6 cells with paclitaxel and different DNA damaging agents, such as cisplatin, MMS (methyl methanesulfonate) and 4NQO (4-nitroquinoline1-oxide), and evaluated cellular survival. We also evaluated DNA damage protein such as gamma-H2AX, CHK1, and CHK2 expression and cell cycle during cisplatin treatment in these two cells. We also checked DNA repair protein expression, such as UBC13, BRCA1, FANCD2, HLTF and SHPRH in HONE-1 and HONE-6 cells by qPCR and Western blotting. By using confocal microscopy and sister chromatin exchange, we evaluated the phenotypes in HONE-1 and HONE-6 cells. Finally, though transfection with shRNA of various DNA repair proteins, we elucidate the biological role of DNA repair in the phenotype of HONE-6 cells, grown under chronic cisplatin treatment. Results and Discussion: Compared to parental HONE-1 cells, HONE-6 cells were more resistant to DNA damage agents as cisplatin, 4NQO, MMS, but not to mitotic inhibitor, paclitaxel. Cisplatin induced more gamma-H2AX expression, CHK1 and CHK2 activation, and cell cycle arrest in S phase in HONE-1 cells than in HONE-6 cells. More gamma-H2AX foci were also found though confocal microscopy in HONE-1 cells than in HONE-6 cells. Therefore, cisplatin induced more DNA damage in HONE-1 cells than in HONE-6 cells. At cellular baseline, there were more DNA repair protein expression, such as BRCA1, FANCD2, UBC13, HLTF and SHPRH in HONE-6 cells than in HONE-1 cells. Besides, cisplatin induced more BRCA1 and FANCD2 expression in HONE-6 cells than in HONE-1 cells. Strikingly compared to HONE-1 cells, HONE6 cells show elevated sister chromatin exchange, which is the direct evidence for DNA repair. Finally in HONE-6 cells, compared to vector control, down-regulation of DNA repair protein by shRNA, such as FANCD2, BRCA1, UBC13 and HLTF (except SHPRH), led to the cancer cells have more cisplatininduced gamma-H2AX foci and made the cancer cells more sensitive to cisplatin. Conclusion: HONE-6 cells, derived from HONE-1 cells and grown under chronic cisplatin treatment, possessed higher ability of DNA repair − both
homologous recombination and template switching, which led to cellular resistance to cisplatin. No conflict of interest. 568 The role of Slit1 in angiogenesis K.F. Pan1 , T.Y. Yeh1 , F.C. Peng1 . 1 Graduate Institute of Toxicology, College of Medicine National Taiwan University, Taipei City, Taiwan Background: Slits are large, secreted repulsive axon guidance molecules. Mammals have 3 Slit proteins (Slit1−3) which bind to Roundabout receptors (Robo1−4) that induce signaling during axon guidance and neuronal migration. Recent studies showed that molecules which played important roles in development were often dysregulated in cancer and some references have reported that both Slit2 and Slit3 proteins promoted angiogenesis, a process essential for tumor growth. But the role of Slit1 in angiogenesis is unclear. Therefore in this study, we investigated the angiogenic effects of Slit1. Materials and Methods: We used primary human umbilical vein endothelial cells (HUVECs) as angiogenesis model. To investigate the expression of Robo1 and Robo4 receptors in HUVECs, immunofluorescence and western blotting were conducted. The tube formation assay, wound healing assay and cell proliferation assay were tested to confirm the angiogenic effects of Slit1. Results: We observed that HUVECs express Robo1 and Robo4 receptors suggesting that Slit1 may interact with Robo1 or Robo4 to regulate endothelial cells functions. To assess whether Slit1 can promote angiogenesis in vitro, HUVECs were treated with recombinant Slit1 proteins and examined by cell proliferation assay, wound healing assay and tube formation assay. We found that Slit1 dose dependently promoted vascular morphogenesis. But did not affect HUVECs proliferation and motility abilities. Furthermore, the tube formation assay was repeated with function blocking Robo4 peptide and the result showed that the interaction between Slit1 and Robo4 disrupted Slit1induced HUVECs tube formation. Conclusion: Previous studies showed that some molecules, for example: angiogenin had no effect on endothelial cell proliferation and migration, but it could still influence the process of angiogenesis, classified as angiogenic activator. Our data demonstrated that Slit1 only had impact on vascular morphogenesis, indicated that Slit1 may function as a novel angiogenic factor. However, the mechanism of Slit1 mediated vascular morphogenesis still need to verify in the future. No conflict of interest. 569 Chronic and low doses exposure of environmental pollutants induces phenotypic alterations in a tumour progression model of breast cancer C.F. Donini1 , E. Grisard2 , V. Maguer-Satta2 , P. Balaguer3 , A. Escande4 , M.L. Bayle5 , C. Casellas4 , V. Cavailles3 , B. Fervers1 , P.A. Cohen1 . 1 ´ ´ Centre Leon Berard, Departement Cancer et Environnement, Lyon, France, 2 CRCL-Inserm U1052-CNRS U5286, Lyon, France, 3 Inserm U896, Montpellier, France, 4 UMR5569 (UMI UMII IRD CNRS), Montpellier, France, 5 Plateforme de Recherche en Toxicologie Environnementale et Ecotoxicologie de Rovaltain, Valence, France Background: Breast cancer is the most common cancer and one of the major causes of cancer deaths in women world-wide. Growing evidence indicates that exposure to environmental carcinogens may increase the risk of sporadic breast cancers, so it is of most importance to decipher the role of environmental carcinogens in the chronic carcinogenesis of human breast epithelia and their contribution in the development of this cancer. In this study we characterized a breast tumour progression model, including a nontumorigenic human mammary epithelial cell line, a premalignant cell line and a fully malignant tumour cell line, in order to validate its suitability for further studies with environmental factors. Two environmental factors possessing distinct mechanisms of toxicity (genotoxicity and endocrine disruption) and representing a real issue among environmental risks were chosen for chronic and low doses exposure of the breast tumour progression model. Material and Methods: A breast tumour progression model that mimics progression from benign to premalignant or fully malignant phenotype was used. To corroborate its appropriateness for environmental chronic and low-doses exposure of environmental pollutants, we performed proliferation assays, western blot analysis and anchorage-independent growth assays. All cell lines were thereafter exposed to low-doses of an endocrine disruptor or a genotoxic compound, or both of them for 60 days. In order to characterize the cellular and molecular phenotypes developed by the exposed cell lines, and to determine whether the combination of both environmental pollutants enhances the effect of each molecules tested individually, we carried out cell growth analysis, investigated the activation of key signalling pathways, and performed anchorage-independent growth assays. Results: Our results demonstrated that the increasing malignant potential of the tumour progression model was associated with increased cell proliferation rate, increased phosphorylation of key signalling proteins and increased anchorage-independent growth. This tumour progression model
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
566 Investigating PI3Kinase and K-ras pathway interactions in mouse model of colorectal cancer V. Meniel1 , I. Martin-Berenjeno2 , B. Vanhaesebroeck2 , A.R. Clarke1 . 1 Cardiff University, European Cancer Stem Cell Research Institute, Cardiff Wales, United Kingdom, 2 University College London, UCL Cancer Institute, London, United Kingdom Introduction: K-Ras, the p110a catalytic subunit of PI3K (PIK3CA), and the negative regulator, PTEN, are all frequently altered in colorectal cancer. Mutation in these genes leads to aberrant activation of PI3K/Akt and MAPK/Erk signaling pathways. 21.3% of colorectal cancers with K-ras or B-raf mutations have concomitant mutations with PI3KCA whereas PTEN loss and PI3KCA mutations have been shown to co-exist in 6.6% colorectal cancers. Previously we have shown that PTEN loss in the mouse leads to increased tumorigenicity in the context of Apc deficiency and similarly activating mutation K-rasV12 accelerated intestinal tumorigenesis and progression. Material and Methods: Using our Cre-lox Apc mouse model, we are now investigating the effect on intestinal tumorigenesis and progression after dual Wnt signalling activation and PI3KCA oncogenic activation using the H1047R hot spot activating mutation (P110aonc). To study the synergy between aberrant activation of PI3K/Akt pathway and MAPK/Erk patwhay, we are also studying the additional effect of K-ras activated mutation or Pten deficiency in the context of PI3KCA activation together with loss of Apc. Results and Discussion: We are currently generating cohorts of mice Flp-P110oncVillinApcfl/+ , Flp-P110aoncVillinApcfl/+ Ptenfl/fl , Flp-P110aoncVillinApcfl/+ KrasV12 together with appropriate controls. Also, we are studying the role of oncogenic activation of PI3KCA in intestinal homeostasis. Preliminary data indicates that tamoxifene induced P110aonc Apcfl/+ mice have a shorter survival compared to tamoxifene induced Apcfl/+ mice. The histopathology, tumour burden and tumour progression analysis of all cohorts are in the process of investigation. No conflict of interest. 567 The impact of chronic cisplatin treatment on DNA repair in head and neck squamous cell carcinoma W. Su1 , W.C. Su1 , J.Y. Chang1 , H.J. Liaw2 . 1 National Cheng Kong University Hospital, Department of Internal Medicine, Tainan City, Taiwan, 2 National Cheng Kong University Hospital, Department of Life Science, Tainan City, Taiwan Introduction: Cisplatin is an important chemotherapeutic agent for many cancers, including head and neck squamous cell carcinoma (HNSCC). Cisplatin cross-links DNA and then interferes with cell division by mitosis. Repeated cisplatin treatment is a way to treat recurrent HNSCC; however, the long-term effect of chronic cisplatin treatment is unknown. We aimed to determine whether chronic cisplatin treatment induced more ability of DNA repair. Material and Methods: Human nasopharyngeal carcinoma cells (HONE-1) and its cisplatin resistant HONE-6 cells were gifts from Prof. Chang JangYang. HONE-6 cells were developed through repeated selection from the parental HONE-1 cells using cisplatin containing medium. We treated HONE-1 and HONE-6 cells with paclitaxel and different DNA damaging agents, such as cisplatin, MMS (methyl methanesulfonate) and 4NQO (4-nitroquinoline1-oxide), and evaluated cellular survival. We also evaluated DNA damage protein such as gamma-H2AX, CHK1, and CHK2 expression and cell cycle during cisplatin treatment in these two cells. We also checked DNA repair protein expression, such as UBC13, BRCA1, FANCD2, HLTF and SHPRH in HONE-1 and HONE-6 cells by qPCR and Western blotting. By using confocal microscopy and sister chromatin exchange, we evaluated the phenotypes in HONE-1 and HONE-6 cells. Finally, though transfection with shRNA of various DNA repair proteins, we elucidate the biological role of DNA repair in the phenotype of HONE-6 cells, grown under chronic cisplatin treatment. Results and Discussion: Compared to parental HONE-1 cells, HONE-6 cells were more resistant to DNA damage agents as cisplatin, 4NQO, MMS, but not to mitotic inhibitor, paclitaxel. Cisplatin induced more gamma-H2AX expression, CHK1 and CHK2 activation, and cell cycle arrest in S phase in HONE-1 cells than in HONE-6 cells. More gamma-H2AX foci were also found though confocal microscopy in HONE-1 cells than in HONE-6 cells. Therefore, cisplatin induced more DNA damage in HONE-1 cells than in HONE-6 cells. At cellular baseline, there were more DNA repair protein expression, such as BRCA1, FANCD2, UBC13, HLTF and SHPRH in HONE-6 cells than in HONE-1 cells. Besides, cisplatin induced more BRCA1 and FANCD2 expression in HONE-6 cells than in HONE-1 cells. Strikingly compared to HONE-1 cells, HONE6 cells show elevated sister chromatin exchange, which is the direct evidence for DNA repair. Finally in HONE-6 cells, compared to vector control, down-regulation of DNA repair protein by shRNA, such as FANCD2, BRCA1, UBC13 and HLTF (except SHPRH), led to the cancer cells have more cisplatininduced gamma-H2AX foci and made the cancer cells more sensitive to cisplatin. Conclusion: HONE-6 cells, derived from HONE-1 cells and grown under chronic cisplatin treatment, possessed higher ability of DNA repair − both
homologous recombination and template switching, which led to cellular resistance to cisplatin. No conflict of interest. 568 The role of Slit1 in angiogenesis K.F. Pan1 , T.Y. Yeh1 , F.C. Peng1 . 1 Graduate Institute of Toxicology, College of Medicine National Taiwan University, Taipei City, Taiwan Background: Slits are large, secreted repulsive axon guidance molecules. Mammals have 3 Slit proteins (Slit1−3) which bind to Roundabout receptors (Robo1−4) that induce signaling during axon guidance and neuronal migration. Recent studies showed that molecules which played important roles in development were often dysregulated in cancer and some references have reported that both Slit2 and Slit3 proteins promoted angiogenesis, a process essential for tumor growth. But the role of Slit1 in angiogenesis is unclear. Therefore in this study, we investigated the angiogenic effects of Slit1. Materials and Methods: We used primary human umbilical vein endothelial cells (HUVECs) as angiogenesis model. To investigate the expression of Robo1 and Robo4 receptors in HUVECs, immunofluorescence and western blotting were conducted. The tube formation assay, wound healing assay and cell proliferation assay were tested to confirm the angiogenic effects of Slit1. Results: We observed that HUVECs express Robo1 and Robo4 receptors suggesting that Slit1 may interact with Robo1 or Robo4 to regulate endothelial cells functions. To assess whether Slit1 can promote angiogenesis in vitro, HUVECs were treated with recombinant Slit1 proteins and examined by cell proliferation assay, wound healing assay and tube formation assay. We found that Slit1 dose dependently promoted vascular morphogenesis. But did not affect HUVECs proliferation and motility abilities. Furthermore, the tube formation assay was repeated with function blocking Robo4 peptide and the result showed that the interaction between Slit1 and Robo4 disrupted Slit1induced HUVECs tube formation. Conclusion: Previous studies showed that some molecules, for example: angiogenin had no effect on endothelial cell proliferation and migration, but it could still influence the process of angiogenesis, classified as angiogenic activator. Our data demonstrated that Slit1 only had impact on vascular morphogenesis, indicated that Slit1 may function as a novel angiogenic factor. However, the mechanism of Slit1 mediated vascular morphogenesis still need to verify in the future. No conflict of interest. 569 Chronic and low doses exposure of environmental pollutants induces phenotypic alterations in a tumour progression model of breast cancer C.F. Donini1 , E. Grisard2 , V. Maguer-Satta2 , P. Balaguer3 , A. Escande4 , M.L. Bayle5 , C. Casellas4 , V. Cavailles3 , B. Fervers1 , P.A. Cohen1 . 1 ´ ´ Centre Leon Berard, Departement Cancer et Environnement, Lyon, France, 2 CRCL-Inserm U1052-CNRS U5286, Lyon, France, 3 Inserm U896, Montpellier, France, 4 UMR5569 (UMI UMII IRD CNRS), Montpellier, France, 5 Plateforme de Recherche en Toxicologie Environnementale et Ecotoxicologie de Rovaltain, Valence, France Background: Breast cancer is the most common cancer and one of the major causes of cancer deaths in women world-wide. Growing evidence indicates that exposure to environmental carcinogens may increase the risk of sporadic breast cancers, so it is of most importance to decipher the role of environmental carcinogens in the chronic carcinogenesis of human breast epithelia and their contribution in the development of this cancer. In this study we characterized a breast tumour progression model, including a nontumorigenic human mammary epithelial cell line, a premalignant cell line and a fully malignant tumour cell line, in order to validate its suitability for further studies with environmental factors. Two environmental factors possessing distinct mechanisms of toxicity (genotoxicity and endocrine disruption) and representing a real issue among environmental risks were chosen for chronic and low doses exposure of the breast tumour progression model. Material and Methods: A breast tumour progression model that mimics progression from benign to premalignant or fully malignant phenotype was used. To corroborate its appropriateness for environmental chronic and low-doses exposure of environmental pollutants, we performed proliferation assays, western blot analysis and anchorage-independent growth assays. All cell lines were thereafter exposed to low-doses of an endocrine disruptor or a genotoxic compound, or both of them for 60 days. In order to characterize the cellular and molecular phenotypes developed by the exposed cell lines, and to determine whether the combination of both environmental pollutants enhances the effect of each molecules tested individually, we carried out cell growth analysis, investigated the activation of key signalling pathways, and performed anchorage-independent growth assays. Results: Our results demonstrated that the increasing malignant potential of the tumour progression model was associated with increased cell proliferation rate, increased phosphorylation of key signalling proteins and increased anchorage-independent growth. This tumour progression model