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915: Claudin-3 overexpression confers radioresistance of colorectal cancer cells
S224
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
outcome of external beam radiotherapy or of brachytherapy. For radiobiological evaluation results of radiotherapy cancer cervix we used the LQ model BED. Material and Method: Were studied the data about 1756 patients with carcinoma of the uterine cervix IB−3B stage, who obtained the radiotherapy in N.N. Petrov Research Institute of Oncology Ministry of Health, SaintPetersburg. For treating all patients was used the combination (EBRT + Brachytherapy) radiotherapy. For EBRT was used the Russian linear accelerators of electrons LUEV-15 M1 (Ex = 15 MeV) and LUE-25 (Ex = 25 MeV) with conventional modes fractionating target dose. Brachytherapy with LDR (0.5 Gy/h) was underwent 235 patients. Three modes were applied for fractionating the summary target dose (STD): 1−20 Gy, once a week, STD = 80 Gy (BEDe = 85, BEDl = 139), 2−20 Gy, once a week, STD = 100 Gy (BEDe = 106, BEDl = 174), 3−20 Gy, once a week, STD = 120 Gy (BEDe = 127, BEDl = 209). Brachytherapy with MDR (5−10 Gy/h) was underwent 542 patients and with HDR (>12 Gy/h) treated 979 patients. Three modes of MDR and HDR brachytherapy were applied for fractionating the summary target dose (STD): 1−7 Gy, once a week, STD = 28 Gy (BEDe = 48, BEDl = 106), 2−7 Gy, once a week, STD = 35 Gy (BEDe = 60, BEDl = 133), 3−7 Gy, once a week, STD = 42 Gy (BEDe = 71, BEDl = 160). For clinical and radiobiological evaluation results of radiotherapy cancer cervix all patients were separated for two groups. In the First group were included patients with radiobiological summary target dose from combine radiotherapy BEDe 85 Gy and BEDl 160 Gy. In the Second group were included patients with BED > 85 Gy and BEDl > 160 Gy. Results: The total number of acute toxicity among patients in the Second group (29%) treated with combination radiotherapy substantially (p < 0.05) was 10% more in comparison with Fist group (19%). The total number of late toxicity among patients in the First group (4%) treated with combination radiotherapy substantially (p < 0.05) was 5% less in comparison with Second group (9%). Conclusion: Quantity early and late toxicity substantially depends on value radiobiological summary target dose for combination radiotherapy carcinoma of the uterine cervix. No conflict of interest. 915 Claudin-3 overexpression confers radioresistance of colorectal cancer cells N. Fortunato-Miranda1 , W.F. Souza1 , J.A. Morgado-D´ıaz1 . 1 Instituto Nacional ˆ de Cancer, Grupo de Biologia Estrutural Programa de Biologia Celular Centro de Pesquisa, Rio de Janeiro, Brazil Background: Colorectal cancer (CRC) represents one of the main causes of cancer-related mortality in the world. During the malignancy process, epithelial cells undergo morphogenetic changes, such as disassembly of cell–cell adhesion system, which leads to the development of epithelial–mesenchymal transition. Tight junctions (TJs) as part of the cell–cell adhesion system, play an important role in this process. Claudins are the main proteins that regulate the functions of TJs, therefor alterations in the expression, stability and subcellular localization of these proteins has been associated with a myriad of diseases, including cancer. Ionizing radiation (IR) is used as first line of treatment for CRC patients. However, the local recurrence, second malignances and metastasis remain a problem of this therapy since irradiatedtissue-microenvironment could modulated the survivor progeny conferring potential malignant advantages. The purpose of this study was to evaluate the tumorigenic potential of the progeny derived from irradiated survivor CRC cell line, that overexpress claudin-3. Material and Methods: In this study we used a cell line derived of human colorectal cancer, HT-29, which was transduced with claudin-3 cDNA using a retroviral vector to overexpress this protein (CLD3) or an empty vector (mock). Cells were irradiated with 5 Gy in a Cs137 irradiator. After 24 h, cells were trypsinized and maintained in culture to form colonies. Cellular events related with the tumorigenic potential such as cell morphology, proliferation, invasiveness, and anchorage independent growth were monitored. Results and Discussion: We demonstrated that survival progeny derived from irradiated parental HT-29 cells (wild type) showed a decreased expression of claudin-3 and that IR did not alter the expression in survival progeny that overexpress this protein as well as presented an aberrant morphology. Furthermore, we observed that non-irradiated CLD3 cells display increased proliferation and that IR has no effect on proliferation of mock cells, however in CLD3 cells, IR leaded to a decreasing of it. In addition, survival progeny of cells overexpressing this protein decreased foci formation as compared with non-irradiated cells. Interestingly, IR increased invasion capacity of irradiated CLD3 cells as compared to irradiated mock cells. Conclusion: The results suggest that survival progeny derived of parental irradiated HT-29 cells that overexpress claudin-3 are more radiorresistant and this indicate an increased aggressive potential, which could contribute to the development of refractory tumors and metastasis. No conflict of interest.
916 Importance of stromal caveolin-1 for tumor growth and radiosensitivity of epithelial tumors V. Verhelst1 , D. Klein1 , T. Schmitz1 , A. Sak2 , V. Jendrossek1 . 1 University Hospital Essen, Institute for Cell Biology (Cancer Research) University of Duisburg-Essen, Essen, Germany, 2 University Hospital Essen, Institute of Radiotherapy (Cancer Research) University of Duisburg-Essen, Essen, Germany Introduction: Resistance of solid tumors to chemotherapy and radiotherapy remains a major obstacle in anticancer treatment. Increasing evidence indicates that the heterogeneous tumor microenvironment supports therapy resistance resulting in poor clinical outcome. However, the mechanisms driving stroma-mediated tumor growth and radiation resistance remain to be defined. Aim of the present study was to investigate the role of stromal caveolin-1 (Cav1) for growth- and resistance-promoting tumor–stroma interactions during tumor progression. Material and Methods: Syngenic tumor cells of different tumor entities (B16F10 melanoma, MPR prostate carcinoma and LL2 Lewis lung adenocarcinoma) were subcutaneously implanted into Cav1-deficient host animals and their wild type littermates (C57BL/6 background). Tumor growth and sensitivity to ionizing radiation were recorded and isolated tumor tissues were analyzed by immunohistochemistry, Westernblot analysis and RealTime PCR. Moreover we performed in vitro studies analyzing the impact of Cav1 deficient stromal cells on tumor cell proliferation, survival and radiation sensitivity. Results and Discussion: Tumors implanted into Cav1-deficient mice grew faster and displayed an enlarged vascular density, a lack of mural cells in tumor vessels and an increased deposition of extracellular matrix molecules in the stroma when compared to tumors grown on wild type animals. Moreover, radiation sensitivity of tumors strongly depended on the tumor entity and stromal Cav1 expression: While the growth delay of LL2 tumors and B16F10 tumors after irradiation was comparable in Cav1 deficient animals and wild type control animals, MPR tumors were more sensitive to radiation when grown on Cav1 deficient hosts compared to MPR tumors grown on respective wild type controls. Interestingly, supernatants of non-irradiated Cav1-deficient fibroblasts promoted the viability of MPR tumor cells in vitro, whereas supernatants of irradiated Cav1-deficient fibroblasts decreased MPR cell viability. Conclusions: Our data suggest a tumor cell specific role of stromal Cav1 for the radiation response of experimental tumors. Present investigations aim to understand the underlying mechanisms by defining differences in the secretome of wild type and Cav1 deficient stroma cells without and with irradiation by a proteomics approach which may result in the identification of novel therapeutic targets for improving clinical outcome. No conflict of interest.
Monday 7 July 2014 Poster Session
Radiobiology/Radiation Oncology II 917 PI3K/Akt and ERK1/2 signaling pathways are involved with the aggressive potential of the progeny derived from radioresistant colorectal cancer cells P.G. Marcondes1 , L.G. Bastos1 , J.A. Morgado-D´ıaz1 . 1 Brazilian National Cancer Institute, Cellular Biology, Rio de Janeiro, Brazil Background: Colorectal cancer (CRC) is one of the most common tumors among population and radiotherapy is used for treatment of rectal cancer. However, irradiated tissues produce a microenvironment that could induce signaling pathways and modulate the radiation-survivor cells, conferring them some malignant advantages. Furthermore, fractionation schedules frequently allows the repopulation of surviving tumor cells and subsequent metastasis. The aim of this study was to analyze the signaling pathways induced by radiation on the progeny derived from irradiation survivor parental cells that may be supporting the aggressive features showed by this progeny. Material and Methods: The CRC cell line HT-29 was used as a model in the experiments. The cells were irradiated with 5 Gy by using a Cs137 irradiator and after 24 hours, cells were trypsinized and maintained in culture to form progeny colonies. Immunoblotting was performed to evaluate protein expression. Cell morphology was analyzed by phase contrast microscopy. The pharmacological inhibitors LY294002 and PD98059 were used to block PI3K/AKT and ERK1/2 signaling pathways, respectively. Transwell with and without Matrigel® coating was used to analyze cell invasiveness and migratory potentials, respectively. Results: The progeny derived from irradiation-survivor cells showed an overexpression of the phosphorylated form of AKT and ERK1/2 kinases compared to the control progeny. Then, pharmacological inhibition of these kinases was used in order to analyze their involvement in the acquisition of the malignant phenotype induced by the radiation. The aberrant cell morphology,
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
outcome of external beam radiotherapy or of brachytherapy. For radiobiological evaluation results of radiotherapy cancer cervix we used the LQ model BED. Material and Method: Were studied the data about 1756 patients with carcinoma of the uterine cervix IB−3B stage, who obtained the radiotherapy in N.N. Petrov Research Institute of Oncology Ministry of Health, SaintPetersburg. For treating all patients was used the combination (EBRT + Brachytherapy) radiotherapy. For EBRT was used the Russian linear accelerators of electrons LUEV-15 M1 (Ex = 15 MeV) and LUE-25 (Ex = 25 MeV) with conventional modes fractionating target dose. Brachytherapy with LDR (0.5 Gy/h) was underwent 235 patients. Three modes were applied for fractionating the summary target dose (STD): 1−20 Gy, once a week, STD = 80 Gy (BEDe = 85, BEDl = 139), 2−20 Gy, once a week, STD = 100 Gy (BEDe = 106, BEDl = 174), 3−20 Gy, once a week, STD = 120 Gy (BEDe = 127, BEDl = 209). Brachytherapy with MDR (5−10 Gy/h) was underwent 542 patients and with HDR (>12 Gy/h) treated 979 patients. Three modes of MDR and HDR brachytherapy were applied for fractionating the summary target dose (STD): 1−7 Gy, once a week, STD = 28 Gy (BEDe = 48, BEDl = 106), 2−7 Gy, once a week, STD = 35 Gy (BEDe = 60, BEDl = 133), 3−7 Gy, once a week, STD = 42 Gy (BEDe = 71, BEDl = 160). For clinical and radiobiological evaluation results of radiotherapy cancer cervix all patients were separated for two groups. In the First group were included patients with radiobiological summary target dose from combine radiotherapy BEDe 85 Gy and BEDl 160 Gy. In the Second group were included patients with BED > 85 Gy and BEDl > 160 Gy. Results: The total number of acute toxicity among patients in the Second group (29%) treated with combination radiotherapy substantially (p < 0.05) was 10% more in comparison with Fist group (19%). The total number of late toxicity among patients in the First group (4%) treated with combination radiotherapy substantially (p < 0.05) was 5% less in comparison with Second group (9%). Conclusion: Quantity early and late toxicity substantially depends on value radiobiological summary target dose for combination radiotherapy carcinoma of the uterine cervix. No conflict of interest. 915 Claudin-3 overexpression confers radioresistance of colorectal cancer cells N. Fortunato-Miranda1 , W.F. Souza1 , J.A. Morgado-D´ıaz1 . 1 Instituto Nacional ˆ de Cancer, Grupo de Biologia Estrutural Programa de Biologia Celular Centro de Pesquisa, Rio de Janeiro, Brazil Background: Colorectal cancer (CRC) represents one of the main causes of cancer-related mortality in the world. During the malignancy process, epithelial cells undergo morphogenetic changes, such as disassembly of cell–cell adhesion system, which leads to the development of epithelial–mesenchymal transition. Tight junctions (TJs) as part of the cell–cell adhesion system, play an important role in this process. Claudins are the main proteins that regulate the functions of TJs, therefor alterations in the expression, stability and subcellular localization of these proteins has been associated with a myriad of diseases, including cancer. Ionizing radiation (IR) is used as first line of treatment for CRC patients. However, the local recurrence, second malignances and metastasis remain a problem of this therapy since irradiatedtissue-microenvironment could modulated the survivor progeny conferring potential malignant advantages. The purpose of this study was to evaluate the tumorigenic potential of the progeny derived from irradiated survivor CRC cell line, that overexpress claudin-3. Material and Methods: In this study we used a cell line derived of human colorectal cancer, HT-29, which was transduced with claudin-3 cDNA using a retroviral vector to overexpress this protein (CLD3) or an empty vector (mock). Cells were irradiated with 5 Gy in a Cs137 irradiator. After 24 h, cells were trypsinized and maintained in culture to form colonies. Cellular events related with the tumorigenic potential such as cell morphology, proliferation, invasiveness, and anchorage independent growth were monitored. Results and Discussion: We demonstrated that survival progeny derived from irradiated parental HT-29 cells (wild type) showed a decreased expression of claudin-3 and that IR did not alter the expression in survival progeny that overexpress this protein as well as presented an aberrant morphology. Furthermore, we observed that non-irradiated CLD3 cells display increased proliferation and that IR has no effect on proliferation of mock cells, however in CLD3 cells, IR leaded to a decreasing of it. In addition, survival progeny of cells overexpressing this protein decreased foci formation as compared with non-irradiated cells. Interestingly, IR increased invasion capacity of irradiated CLD3 cells as compared to irradiated mock cells. Conclusion: The results suggest that survival progeny derived of parental irradiated HT-29 cells that overexpress claudin-3 are more radiorresistant and this indicate an increased aggressive potential, which could contribute to the development of refractory tumors and metastasis. No conflict of interest.
916 Importance of stromal caveolin-1 for tumor growth and radiosensitivity of epithelial tumors V. Verhelst1 , D. Klein1 , T. Schmitz1 , A. Sak2 , V. Jendrossek1 . 1 University Hospital Essen, Institute for Cell Biology (Cancer Research) University of Duisburg-Essen, Essen, Germany, 2 University Hospital Essen, Institute of Radiotherapy (Cancer Research) University of Duisburg-Essen, Essen, Germany Introduction: Resistance of solid tumors to chemotherapy and radiotherapy remains a major obstacle in anticancer treatment. Increasing evidence indicates that the heterogeneous tumor microenvironment supports therapy resistance resulting in poor clinical outcome. However, the mechanisms driving stroma-mediated tumor growth and radiation resistance remain to be defined. Aim of the present study was to investigate the role of stromal caveolin-1 (Cav1) for growth- and resistance-promoting tumor–stroma interactions during tumor progression. Material and Methods: Syngenic tumor cells of different tumor entities (B16F10 melanoma, MPR prostate carcinoma and LL2 Lewis lung adenocarcinoma) were subcutaneously implanted into Cav1-deficient host animals and their wild type littermates (C57BL/6 background). Tumor growth and sensitivity to ionizing radiation were recorded and isolated tumor tissues were analyzed by immunohistochemistry, Westernblot analysis and RealTime PCR. Moreover we performed in vitro studies analyzing the impact of Cav1 deficient stromal cells on tumor cell proliferation, survival and radiation sensitivity. Results and Discussion: Tumors implanted into Cav1-deficient mice grew faster and displayed an enlarged vascular density, a lack of mural cells in tumor vessels and an increased deposition of extracellular matrix molecules in the stroma when compared to tumors grown on wild type animals. Moreover, radiation sensitivity of tumors strongly depended on the tumor entity and stromal Cav1 expression: While the growth delay of LL2 tumors and B16F10 tumors after irradiation was comparable in Cav1 deficient animals and wild type control animals, MPR tumors were more sensitive to radiation when grown on Cav1 deficient hosts compared to MPR tumors grown on respective wild type controls. Interestingly, supernatants of non-irradiated Cav1-deficient fibroblasts promoted the viability of MPR tumor cells in vitro, whereas supernatants of irradiated Cav1-deficient fibroblasts decreased MPR cell viability. Conclusions: Our data suggest a tumor cell specific role of stromal Cav1 for the radiation response of experimental tumors. Present investigations aim to understand the underlying mechanisms by defining differences in the secretome of wild type and Cav1 deficient stroma cells without and with irradiation by a proteomics approach which may result in the identification of novel therapeutic targets for improving clinical outcome. No conflict of interest.
Monday 7 July 2014 Poster Session
Radiobiology/Radiation Oncology II 917 PI3K/Akt and ERK1/2 signaling pathways are involved with the aggressive potential of the progeny derived from radioresistant colorectal cancer cells P.G. Marcondes1 , L.G. Bastos1 , J.A. Morgado-D´ıaz1 . 1 Brazilian National Cancer Institute, Cellular Biology, Rio de Janeiro, Brazil Background: Colorectal cancer (CRC) is one of the most common tumors among population and radiotherapy is used for treatment of rectal cancer. However, irradiated tissues produce a microenvironment that could induce signaling pathways and modulate the radiation-survivor cells, conferring them some malignant advantages. Furthermore, fractionation schedules frequently allows the repopulation of surviving tumor cells and subsequent metastasis. The aim of this study was to analyze the signaling pathways induced by radiation on the progeny derived from irradiation survivor parental cells that may be supporting the aggressive features showed by this progeny. Material and Methods: The CRC cell line HT-29 was used as a model in the experiments. The cells were irradiated with 5 Gy by using a Cs137 irradiator and after 24 hours, cells were trypsinized and maintained in culture to form progeny colonies. Immunoblotting was performed to evaluate protein expression. Cell morphology was analyzed by phase contrast microscopy. The pharmacological inhibitors LY294002 and PD98059 were used to block PI3K/AKT and ERK1/2 signaling pathways, respectively. Transwell with and without Matrigel® coating was used to analyze cell invasiveness and migratory potentials, respectively. Results: The progeny derived from irradiation-survivor cells showed an overexpression of the phosphorylated form of AKT and ERK1/2 kinases compared to the control progeny. Then, pharmacological inhibition of these kinases was used in order to analyze their involvement in the acquisition of the malignant phenotype induced by the radiation. The aberrant cell morphology,