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Autophago-Lysosomal Flux Following Exposure of Endothelial Cells and Fibroblasts to Ionizing Radiation
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Volume 90 Number 1S Supplement 2014 miR-125b, and prevented miR-125b-mediated myeloid expansion in vitro. We then tested the effect of IRF4 (linked to GFP fluorophore) and miR-125b (linked to mCherry fluorophore) co-expression in the mouse hematopoietic system via retroviral transduction and bone marrow transplantation. Stunningly, the in vivo hematopoietic compartment over-expressing both miR-125b and IRF4 had normal numbers of myeloid cells and was similar to controls, in stark contrast to the myeloid hyperproliferation seen in cells over-expressing only miR-125b (1x10^6 myeloid cells/ml vs 12x10^6 myeloid cells/ml, p<0.001). Moreover, none of the mice who overexpressed both miR-125b and IRF4 developed myeloid leukemia, compared to 90% of mice overexpressing only miR-125b. Conclusions: MiR-125b is an oncogenic microRNA that induces an aggressive myeloid leukemia in mice via inhibition of the transcription factor IRF4 in hematopoietic cells. Inhibiting this oncogenic microRNA via targeted overexpression of IRF4 may prove to be highly therapeutic in certain forms of mammalian myeloid leukemia. Author Disclosure: A. Chaudhuri: None. A.Y. So: None. R. Sookram: None. D. Baltimore: K. Advisory Board; Regulus Therapeutics.
3423 Autophago-Lysosomal Flux Following Exposure of Endothelial Cells and Fibroblasts to Ionizing Radiation M.I. Koukourakis, D. Kalamida, I. Karagounis, and A. Giatromanolaki; Democritus University of Thrace, Medical School, Alexandroupolis, Greece Purpose/Objective(s): Vasculature and connective tissue damage is an important contributor of radiation therapy side-effects. The aim of this study is to provide insights in the radiobiology of the autophagic response of endothelial cells and fibroblasts. Materials/Methods: Human vascular endothelial cells (HuVEC) and MRC5 fibroblasts were exposed to 2Gy of ionizing radiation (IR) and an autophagic characterization was performed by confocal microscopy and Western blot analysis, at 4 and 8 days post-irradiation using a variety of antibodies for MAP1LC3A and B, LAMP2A, p62 and CathepsinD. Cell proliferation and survival experiments was performed using the AlamarBlueÒ assay. LC3A and LC3B expression was suppressed using siRNAs. Results: IR induced accumulation of LC3A+, LC3B+ cytoplasmic vacuoles. The p62/sequstrosome 1 protein was also accumulated suggesting a suppression of the autophago-lysosomal digestive activity. In double immunostaining with lysosomal markers (LAMP2a and CathepsinD) reduction of the colocalization with LC3+ autophagosomes was recorded, suggesting blockage of the “auto-lysosomal flux.” Suppression of LC3A/LC3B proteins with siRNAs resulted in radio-sensitization of both cell lines. Conclusions: The current data provide evidence that autophago-lysosomal activity in endothelial cells and fibroblasts is suppressed by therapeutic doses of IR and plays an important role in radiation-induced cell damage and eventually the development of tissue toxicities. Author Disclosure: M.I. Koukourakis: None. D. Kalamida: None. I. Karagounis: None. A. Giatromanolaki: None.
3424 How Can We Overcome Hypoxia- and Radiation-Induced EMT? Contribution of Blockade on HIF-1 and JNK Phospholyration With LW6 M. Sato, K. Hirose, M. Aoki, Y. Hatayama, H. Kawaguchi, H. Akimoto, Y. Narita, and Y. Takai; Hirosaki University Graduate School of Medicine, Hirosaki, Japan Purpose/Objective(s): Intratumoral hypoxic environment not only contributes to the acquisition of radioresistance but also metastasis. Moreover, it is understood that the surviving cells with radioresistance acquires cell migratory and invasive capabilities through epithelial-mesenchymal transition (EMT) induced by radiation. Suppressing these mechanisms that promote metastasis and relapse after radiation therapy becomes a new problem. This study aimed to analyze the process that each of hypoxia and radiation induce EMT in cancer cells, and to confirm whether LW6, a drug
which has inhibitory effect on HIF-1 through improving intracellular oxygen condition, can inhibits these EMT changes. Materials/Methods: Human non-small cell lung cancer A549 cells were used in this study. Cell motility was evaluated by wound healing assay. Cells were incubated under normoxia (21% O2) or hypoxia (1% O2) for 2 h, then reoxygenated and scratched. After 24 or 48 h, the distance of wounding area and the number of migrated cells were evaluated. For investigating the inhibitory effect on cell motility, cells were treated with LW6, YC-1 as a HIF-1 inhibitor, and MAPK inhibitors such as SP600125 as a JNK inhibitor and SB203580 as a p38 MAPK inhibitor 2 h before hypoxic exposure. Next, cells were X-irradiated with 10 Gy (1 Gy/min), and scratched just after irradiation. Then, cell motility was also evaluated after 24 and 48 h. The cells exposed to hypoxia or irradiation were harvested after 6 h, and protein expression of HIF-1a, p38 and JNK as MAPKs, and N-Cadherin, E-Cadherin and Vimentin as EMT markers were evaluated by Western blot analysis. Results: Each of hypoxia and irradiation increased wound healing activity. LW6 delayed wound healing significantly, compared to untreated cells in each of hypoxia (P<0.01) and irradiated condition (P<0.01), respectively. And YC1 also similarly inhibited wound healing in each conditions (P<0.01, P<0.01). In the cells exposed to hypoxia and irradiation, HIF-1a expression in nuclear fraction as well as phosphorylation of JNK were increased. Regarding to expression of protein as markers of EMT, N-cadherin expression was increased in both of hypoxic and irradiated cells, while upregulation of Vimentin and downregulation of E-cadherin were not observed. Treatment with LW6 strongly inhibited phosphorylation of JNK and slightly inhibited HIF-1a expression. Also, the expression of N-cadherin induced by hypoxic exposure or irradiation was reversed to the original level. Conclusions: The results of this study suggest that both of HIF-1 and JNK activation are potentially involved in the process of EMT induced by each of hypoxia and radiation. Patients with a certain kind of cancer compatible with hypoxia may benefit from a combined therapy of radiation with inhibitors of HIF-1 and JNK activation, such as LW6. Author Disclosure: M. Sato: None. K. Hirose: None. M. Aoki: None. Y. Hatayama: None. H. Kawaguchi: None. H. Akimoto: None. Y. Narita: None. Y. Takai: None.
3425 Normal Tissue Irradiation Promotes Tumor Cell Migration M. Rafat, M. Vilalta Colomer, A.J. Giaccia, and E.E. Graves; Stanford University, Stanford, CA Purpose/Objective(s): Despite aggressive surgical, chemotherapeutic, and radiological intervention, recurrence rates remain high for triplenegative breast cancers. We have previously observed that radiation enhances and modulates the migration of tumor cells in a preclinical breast cancer model. In this work, we characterized the effects of irradiation of normal tissue on tumor cell migration to evaluate how tumor-stromal interactions may influence recurrence after therapy. Materials/Methods: We investigated the ability of media from irradiated normal cells to induce tumor cell invasion in vitro using a transwell assay. Mouse embryonic fibroblasts (MEF) were irradiated at 20 Gy with a cesium source. Supernatant was collected after 2 or 7 d incubation and used as a chemoattractant in an invasion assay performed with 4T1 murine mammary carcinoma cells. In addition, an orthotopic breast cancer model was used to determine the effect of radiation on tumor cell migration to normal tissues. 4T1 cells were stably transduced with viral vectors encoding luciferase and tdTomato for in vivo and ex vivo fluorescence and bioluminescence imaging (BLI). Nude mice were inoculated with 4T1 cells in the mammary fat pad (MFP) and injected with phosphate buffered saline in the contralateral MFP. When tumors reached 7 mm, the contralateral normal MFP was irradiated to a dose of 20 Gy with a 250 kVp cabinet x-ray machine. Cell migration was monitored with BLI 25 days after inoculation. Irradiated tissues were fixed and embedded in paraffin for immunohistochemistry (IHC) analysis. Tissue sections were stained with F4/80 to determine macrophage infiltration. We also visualized tumor cell migration in vivo using intravital microscopy (IVM) by implanting a dorsal skinfold chamber on the back of a mouse. Following tumor
Volume 90 Number 1S Supplement 2014 miR-125b, and prevented miR-125b-mediated myeloid expansion in vitro. We then tested the effect of IRF4 (linked to GFP fluorophore) and miR-125b (linked to mCherry fluorophore) co-expression in the mouse hematopoietic system via retroviral transduction and bone marrow transplantation. Stunningly, the in vivo hematopoietic compartment over-expressing both miR-125b and IRF4 had normal numbers of myeloid cells and was similar to controls, in stark contrast to the myeloid hyperproliferation seen in cells over-expressing only miR-125b (1x10^6 myeloid cells/ml vs 12x10^6 myeloid cells/ml, p<0.001). Moreover, none of the mice who overexpressed both miR-125b and IRF4 developed myeloid leukemia, compared to 90% of mice overexpressing only miR-125b. Conclusions: MiR-125b is an oncogenic microRNA that induces an aggressive myeloid leukemia in mice via inhibition of the transcription factor IRF4 in hematopoietic cells. Inhibiting this oncogenic microRNA via targeted overexpression of IRF4 may prove to be highly therapeutic in certain forms of mammalian myeloid leukemia. Author Disclosure: A. Chaudhuri: None. A.Y. So: None. R. Sookram: None. D. Baltimore: K. Advisory Board; Regulus Therapeutics.
3423 Autophago-Lysosomal Flux Following Exposure of Endothelial Cells and Fibroblasts to Ionizing Radiation M.I. Koukourakis, D. Kalamida, I. Karagounis, and A. Giatromanolaki; Democritus University of Thrace, Medical School, Alexandroupolis, Greece Purpose/Objective(s): Vasculature and connective tissue damage is an important contributor of radiation therapy side-effects. The aim of this study is to provide insights in the radiobiology of the autophagic response of endothelial cells and fibroblasts. Materials/Methods: Human vascular endothelial cells (HuVEC) and MRC5 fibroblasts were exposed to 2Gy of ionizing radiation (IR) and an autophagic characterization was performed by confocal microscopy and Western blot analysis, at 4 and 8 days post-irradiation using a variety of antibodies for MAP1LC3A and B, LAMP2A, p62 and CathepsinD. Cell proliferation and survival experiments was performed using the AlamarBlueÒ assay. LC3A and LC3B expression was suppressed using siRNAs. Results: IR induced accumulation of LC3A+, LC3B+ cytoplasmic vacuoles. The p62/sequstrosome 1 protein was also accumulated suggesting a suppression of the autophago-lysosomal digestive activity. In double immunostaining with lysosomal markers (LAMP2a and CathepsinD) reduction of the colocalization with LC3+ autophagosomes was recorded, suggesting blockage of the “auto-lysosomal flux.” Suppression of LC3A/LC3B proteins with siRNAs resulted in radio-sensitization of both cell lines. Conclusions: The current data provide evidence that autophago-lysosomal activity in endothelial cells and fibroblasts is suppressed by therapeutic doses of IR and plays an important role in radiation-induced cell damage and eventually the development of tissue toxicities. Author Disclosure: M.I. Koukourakis: None. D. Kalamida: None. I. Karagounis: None. A. Giatromanolaki: None.
3424 How Can We Overcome Hypoxia- and Radiation-Induced EMT? Contribution of Blockade on HIF-1 and JNK Phospholyration With LW6 M. Sato, K. Hirose, M. Aoki, Y. Hatayama, H. Kawaguchi, H. Akimoto, Y. Narita, and Y. Takai; Hirosaki University Graduate School of Medicine, Hirosaki, Japan Purpose/Objective(s): Intratumoral hypoxic environment not only contributes to the acquisition of radioresistance but also metastasis. Moreover, it is understood that the surviving cells with radioresistance acquires cell migratory and invasive capabilities through epithelial-mesenchymal transition (EMT) induced by radiation. Suppressing these mechanisms that promote metastasis and relapse after radiation therapy becomes a new problem. This study aimed to analyze the process that each of hypoxia and radiation induce EMT in cancer cells, and to confirm whether LW6, a drug
which has inhibitory effect on HIF-1 through improving intracellular oxygen condition, can inhibits these EMT changes. Materials/Methods: Human non-small cell lung cancer A549 cells were used in this study. Cell motility was evaluated by wound healing assay. Cells were incubated under normoxia (21% O2) or hypoxia (1% O2) for 2 h, then reoxygenated and scratched. After 24 or 48 h, the distance of wounding area and the number of migrated cells were evaluated. For investigating the inhibitory effect on cell motility, cells were treated with LW6, YC-1 as a HIF-1 inhibitor, and MAPK inhibitors such as SP600125 as a JNK inhibitor and SB203580 as a p38 MAPK inhibitor 2 h before hypoxic exposure. Next, cells were X-irradiated with 10 Gy (1 Gy/min), and scratched just after irradiation. Then, cell motility was also evaluated after 24 and 48 h. The cells exposed to hypoxia or irradiation were harvested after 6 h, and protein expression of HIF-1a, p38 and JNK as MAPKs, and N-Cadherin, E-Cadherin and Vimentin as EMT markers were evaluated by Western blot analysis. Results: Each of hypoxia and irradiation increased wound healing activity. LW6 delayed wound healing significantly, compared to untreated cells in each of hypoxia (P<0.01) and irradiated condition (P<0.01), respectively. And YC1 also similarly inhibited wound healing in each conditions (P<0.01, P<0.01). In the cells exposed to hypoxia and irradiation, HIF-1a expression in nuclear fraction as well as phosphorylation of JNK were increased. Regarding to expression of protein as markers of EMT, N-cadherin expression was increased in both of hypoxic and irradiated cells, while upregulation of Vimentin and downregulation of E-cadherin were not observed. Treatment with LW6 strongly inhibited phosphorylation of JNK and slightly inhibited HIF-1a expression. Also, the expression of N-cadherin induced by hypoxic exposure or irradiation was reversed to the original level. Conclusions: The results of this study suggest that both of HIF-1 and JNK activation are potentially involved in the process of EMT induced by each of hypoxia and radiation. Patients with a certain kind of cancer compatible with hypoxia may benefit from a combined therapy of radiation with inhibitors of HIF-1 and JNK activation, such as LW6. Author Disclosure: M. Sato: None. K. Hirose: None. M. Aoki: None. Y. Hatayama: None. H. Kawaguchi: None. H. Akimoto: None. Y. Narita: None. Y. Takai: None.
3425 Normal Tissue Irradiation Promotes Tumor Cell Migration M. Rafat, M. Vilalta Colomer, A.J. Giaccia, and E.E. Graves; Stanford University, Stanford, CA Purpose/Objective(s): Despite aggressive surgical, chemotherapeutic, and radiological intervention, recurrence rates remain high for triplenegative breast cancers. We have previously observed that radiation enhances and modulates the migration of tumor cells in a preclinical breast cancer model. In this work, we characterized the effects of irradiation of normal tissue on tumor cell migration to evaluate how tumor-stromal interactions may influence recurrence after therapy. Materials/Methods: We investigated the ability of media from irradiated normal cells to induce tumor cell invasion in vitro using a transwell assay. Mouse embryonic fibroblasts (MEF) were irradiated at 20 Gy with a cesium source. Supernatant was collected after 2 or 7 d incubation and used as a chemoattractant in an invasion assay performed with 4T1 murine mammary carcinoma cells. In addition, an orthotopic breast cancer model was used to determine the effect of radiation on tumor cell migration to normal tissues. 4T1 cells were stably transduced with viral vectors encoding luciferase and tdTomato for in vivo and ex vivo fluorescence and bioluminescence imaging (BLI). Nude mice were inoculated with 4T1 cells in the mammary fat pad (MFP) and injected with phosphate buffered saline in the contralateral MFP. When tumors reached 7 mm, the contralateral normal MFP was irradiated to a dose of 20 Gy with a 250 kVp cabinet x-ray machine. Cell migration was monitored with BLI 25 days after inoculation. Irradiated tissues were fixed and embedded in paraffin for immunohistochemistry (IHC) analysis. Tissue sections were stained with F4/80 to determine macrophage infiltration. We also visualized tumor cell migration in vivo using intravital microscopy (IVM) by implanting a dorsal skinfold chamber on the back of a mouse. Following tumor