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Thrombin induces pancreatic cancer cell survival during growth factor deprivation
Abstracts / Thrombosis Research 129, Supplement 1 (2012) S155–S194
intense neovascularization. Several lines of evidence indicate that blood clotting enzymes play an important role in the tumor microenvironment, mainly through the activation of protease-activated receptors (PAR). In particular, PAR1 and PAR2 isoforms may activate signal transduction pathways that promote a number of protumoral responses. However, little is known concerning the role of PAR1/PAR2 in GBM progression. In this study, we investigated the expression and function of PAR1 and PAR2 in the human GBM cell lines A172 and U87-MG. We also evaluated the effect of agonist peptides for PAR1 (PAR1-AP) and PAR2 (PAR2-AP) on signaling pathways and the expression of vascular endothelial growth factor (VEGF). Immunoblotting assays showed that A172 and U87-MG constitutively express PAR1 and PAR2. Treatment of GBM cells with PAR1-AP or PAR2-AP enhanced extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt (protein kinase B) phosphorylation in a time-dependent manner. PD98059 and LY29042, inhibitors of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways, decreased PAR-mediated activation of ERK1/2 and Akt, respectively. In addition, we observed that PAR2, but not PAR1, activation increased VEGF secretion in U87-MG and A172 cells. Notably, PD98059, but not LY29042, reduced PAR2-mediated VEGF production by GBM cells. Our results suggest that PAR2 modulates VEGF production through the MAPK/ERK1/2 pathway, and not the PI3K/Akt pathway, in human GBM cell lines. Therefore, the PAR2/MAPK signaling axis might be regarded as a relevant target for adjuvant treatment of GBM with a possible impact on tumor angiogenesis.
PO-80 PAR-2 activation induces migration but not proliferation of pancreatic cancer cells K. Shi, K.C.S. Queiroz, C.A. Spek Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, the Netherlands Introduction: Protease-activated receptor 2 (PAR-2) is a G protein-coupled receptor that functions as a cell-surface sensor for coagulation factors, as well as other proteases associated with the tumor microenvironment. The activation of PAR-2 has been shown to induce proliferation of many cancer cells whereas it also may induce migration. The role of PAR-2 in pancreatic cancer remains largely unexplored however. Aim: The aim of the present study is to examine whether PAR-2 activation induces proliferation and or migration of pancreatic cancer cells and to elucidate the underlying mechanism. Materials and methods: BxPC-3 and Capan-2 pancreatic cancer cell lines were stimulated with PAR-2 agonist peptide or trypsin after which proliferation was assessed by BrdU assays and migration was assessed by trans-well and scratch assays. To identify the underlying signal transduction pathways, the phosphorylation status of Erk1/2 and Src was determined by Western blot analysis. Moreover, specific inhibitors of the different signal transduction pathways were tested in the migration experiments. Results: PAR-2 activation does not induce proliferation of both pancreatic cancer cell lines tested. Interestingly, cell stimulations with PAR-2 agonist peptide and trypsin did induce migration of the pancreatic cancer cells in scratch assays but not in a trans-well setting. Addition of inhibitors of PKC (Chelerythrine), MEK (U0126), Rac (NSC23766) and Src (Dasatinib) all inhibit PAR-2 dependent migration whereas these inhibitors do not inhibit FCS driven migration. In line with these findings, stimulation of BxPC3 and Capan-2 cells does induce phosphorylation of ERK1/2 and Src. Preliminary data suggest that the seeming discrepancy between scratch assays and trans-well experiments may be due to increased metabolic activity of cells in close proximity of the scratch. Ongoing experiments should however elucidate the interplay between PAR-2 and metabolic activity with respect to cancer cell migration. Conclusion: We show that activation of PAR-2 has no effect on proliferation of pancreatic cancer cells but significantly induces migration in scratch assays. We suggest activation of the PKC/Src/Rac/MEK pathway to drive PAR-2 induced pancreatic cancer cell migration but ongoing experiments should confirm this notion. Overall our data suggest that PAR-2 may be important for pancreatic cancer metastasis in an ERK dependent manner.
S191
PO-81 Thrombin induces pancreatic cancer cell survival during growth factor deprivation K.C.S. Queiroz, T. Liechtenstein, C.A. Spek Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, The Netherlands Introduction: Pancreatic cancer patients present an overall 5-year survival rate of less than 5%. This high mortality rate is due to late diagnosis, rapid progression, and resistance to chemotherapy. Pancreatic cancer is often accompanied by ongoing coagulation activation as evident from elevated markers of thrombin generation such as thrombin-anti-thrombin and prothrombin activation fragment F1.2 in patients and anticoagulant treatment may prolong overall survival in patients with advanced pancreatic carcinoma. Hypothesis and aim: Considering the apparent important role of coagulation in pancreatic cancer progression, we hypothesized that thrombindriven signalling could trigger important survival signals in pancreatic cancer cells allowing them to survive in conditions of metabolic stress. Methods: We assessed whether thrombin could protect Panc-1 pancreatic cancer cells from growth factor deprivation-induced cell death. Moreover, we evaluated the underlying signalling pathways triggered by thrombin and used inhibitor studies to validate the importance of the identified pathways. Results: Growth factor deprivation drives Panc1 cells into apoptosis and hardly any colonies are observed in the absence of thrombin. At low thrombin concentration (0.1 U/ml), the number of Panc1 colonies increased significantly, whereas with 0.5 U/ml thrombin the number of colonies only slightly increased and with 1 U/ml we did not see any effect of thrombin. Importantly, thrombin did not affect growth factor deprivation-induced cell death in PAR-1 low BxPC3 pancreatic cancer cells. Thrombin induced phosphorylation of mTOR and its downstream target 4E-BP1, of ERK1/2 and of Akt (which may subsequently lead to sustained mTOR activation). In line with an important role of mTOR in thrombin-driven cancer cell survival under metabolic stress, rapamycin (mTOR inhibitor) prevented thrombin induced cell survival. Interestingly, thrombin also induced AMPK activation, which is intriguing considering that AMPK is most known to down-regulate mTOR. This suggests that thrombin may uncouple AMPK from the mTOR pathway, or alternatively AMPK activation may induce the rictor-complex of mTOR thereby inducing Akt phosphorylation with subsequent sustained mTOR activity. Ongoing experiments aim at discriminating between these two explanations. High thrombin levels (1 U/ml) induced similar phosphorylation events as low thrombin levels although it did induce higher and more sustained activation of ERK. Preliminary data suggest that sustained ERK activity may explain why high thrombin levels do not rescue Panc1 cells form growth factor deprivation-induced cell death. Indeed, in the presence of an ERK inhibitor, high thrombin levels also rescue cells from growth factor deprivation induced cell death. Conclusion: Thrombin protects Panc1 cells in a dose dependent manner from growth factor deprivation-induced cell death and this seems to be dependent on activation of the mTOR pathway.
PO-82 Hypoxia induces Smad2 phosphorylation signaling and increases stem cell-like properties and multi-drug resistance in K562 leukemic cells X.-Y. Cui 1,2 , G. Skretting 1 , Y. Jing 3 , C.F. Myklebust 1 , Y.-F. Liu 2 , P.M. Sandset 1,4 , L. Sun 2 1 Department of Haematology, Oslo University Hospital, Norway; 2 Department of Hematology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China; 3 Department of Physiology, Zhengzhou University Stem Cell Center, Zhengzhou University, Zhengzhou, China; 4 Institute of Clinical Medicine, University of Oslo, Norway Background: Both leukemia and treatment of the disease with chemotherapy can cause hypercoagulability. In general, chemotherapy is an effecient non-targeted therapy for killing the majority of cells in leukemia. However, leukemia cells resistant to such therapy persist in a hypoxic bone marrow niche after chemotherapy, ultimately resulting in patient relapse. Aim: The aim of the present study was to investigate the potential role of hypoxia in maintaining the stem cell-like properties and therapeu-
intense neovascularization. Several lines of evidence indicate that blood clotting enzymes play an important role in the tumor microenvironment, mainly through the activation of protease-activated receptors (PAR). In particular, PAR1 and PAR2 isoforms may activate signal transduction pathways that promote a number of protumoral responses. However, little is known concerning the role of PAR1/PAR2 in GBM progression. In this study, we investigated the expression and function of PAR1 and PAR2 in the human GBM cell lines A172 and U87-MG. We also evaluated the effect of agonist peptides for PAR1 (PAR1-AP) and PAR2 (PAR2-AP) on signaling pathways and the expression of vascular endothelial growth factor (VEGF). Immunoblotting assays showed that A172 and U87-MG constitutively express PAR1 and PAR2. Treatment of GBM cells with PAR1-AP or PAR2-AP enhanced extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt (protein kinase B) phosphorylation in a time-dependent manner. PD98059 and LY29042, inhibitors of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways, decreased PAR-mediated activation of ERK1/2 and Akt, respectively. In addition, we observed that PAR2, but not PAR1, activation increased VEGF secretion in U87-MG and A172 cells. Notably, PD98059, but not LY29042, reduced PAR2-mediated VEGF production by GBM cells. Our results suggest that PAR2 modulates VEGF production through the MAPK/ERK1/2 pathway, and not the PI3K/Akt pathway, in human GBM cell lines. Therefore, the PAR2/MAPK signaling axis might be regarded as a relevant target for adjuvant treatment of GBM with a possible impact on tumor angiogenesis.
PO-80 PAR-2 activation induces migration but not proliferation of pancreatic cancer cells K. Shi, K.C.S. Queiroz, C.A. Spek Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, the Netherlands Introduction: Protease-activated receptor 2 (PAR-2) is a G protein-coupled receptor that functions as a cell-surface sensor for coagulation factors, as well as other proteases associated with the tumor microenvironment. The activation of PAR-2 has been shown to induce proliferation of many cancer cells whereas it also may induce migration. The role of PAR-2 in pancreatic cancer remains largely unexplored however. Aim: The aim of the present study is to examine whether PAR-2 activation induces proliferation and or migration of pancreatic cancer cells and to elucidate the underlying mechanism. Materials and methods: BxPC-3 and Capan-2 pancreatic cancer cell lines were stimulated with PAR-2 agonist peptide or trypsin after which proliferation was assessed by BrdU assays and migration was assessed by trans-well and scratch assays. To identify the underlying signal transduction pathways, the phosphorylation status of Erk1/2 and Src was determined by Western blot analysis. Moreover, specific inhibitors of the different signal transduction pathways were tested in the migration experiments. Results: PAR-2 activation does not induce proliferation of both pancreatic cancer cell lines tested. Interestingly, cell stimulations with PAR-2 agonist peptide and trypsin did induce migration of the pancreatic cancer cells in scratch assays but not in a trans-well setting. Addition of inhibitors of PKC (Chelerythrine), MEK (U0126), Rac (NSC23766) and Src (Dasatinib) all inhibit PAR-2 dependent migration whereas these inhibitors do not inhibit FCS driven migration. In line with these findings, stimulation of BxPC3 and Capan-2 cells does induce phosphorylation of ERK1/2 and Src. Preliminary data suggest that the seeming discrepancy between scratch assays and trans-well experiments may be due to increased metabolic activity of cells in close proximity of the scratch. Ongoing experiments should however elucidate the interplay between PAR-2 and metabolic activity with respect to cancer cell migration. Conclusion: We show that activation of PAR-2 has no effect on proliferation of pancreatic cancer cells but significantly induces migration in scratch assays. We suggest activation of the PKC/Src/Rac/MEK pathway to drive PAR-2 induced pancreatic cancer cell migration but ongoing experiments should confirm this notion. Overall our data suggest that PAR-2 may be important for pancreatic cancer metastasis in an ERK dependent manner.
S191
PO-81 Thrombin induces pancreatic cancer cell survival during growth factor deprivation K.C.S. Queiroz, T. Liechtenstein, C.A. Spek Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, The Netherlands Introduction: Pancreatic cancer patients present an overall 5-year survival rate of less than 5%. This high mortality rate is due to late diagnosis, rapid progression, and resistance to chemotherapy. Pancreatic cancer is often accompanied by ongoing coagulation activation as evident from elevated markers of thrombin generation such as thrombin-anti-thrombin and prothrombin activation fragment F1.2 in patients and anticoagulant treatment may prolong overall survival in patients with advanced pancreatic carcinoma. Hypothesis and aim: Considering the apparent important role of coagulation in pancreatic cancer progression, we hypothesized that thrombindriven signalling could trigger important survival signals in pancreatic cancer cells allowing them to survive in conditions of metabolic stress. Methods: We assessed whether thrombin could protect Panc-1 pancreatic cancer cells from growth factor deprivation-induced cell death. Moreover, we evaluated the underlying signalling pathways triggered by thrombin and used inhibitor studies to validate the importance of the identified pathways. Results: Growth factor deprivation drives Panc1 cells into apoptosis and hardly any colonies are observed in the absence of thrombin. At low thrombin concentration (0.1 U/ml), the number of Panc1 colonies increased significantly, whereas with 0.5 U/ml thrombin the number of colonies only slightly increased and with 1 U/ml we did not see any effect of thrombin. Importantly, thrombin did not affect growth factor deprivation-induced cell death in PAR-1 low BxPC3 pancreatic cancer cells. Thrombin induced phosphorylation of mTOR and its downstream target 4E-BP1, of ERK1/2 and of Akt (which may subsequently lead to sustained mTOR activation). In line with an important role of mTOR in thrombin-driven cancer cell survival under metabolic stress, rapamycin (mTOR inhibitor) prevented thrombin induced cell survival. Interestingly, thrombin also induced AMPK activation, which is intriguing considering that AMPK is most known to down-regulate mTOR. This suggests that thrombin may uncouple AMPK from the mTOR pathway, or alternatively AMPK activation may induce the rictor-complex of mTOR thereby inducing Akt phosphorylation with subsequent sustained mTOR activity. Ongoing experiments aim at discriminating between these two explanations. High thrombin levels (1 U/ml) induced similar phosphorylation events as low thrombin levels although it did induce higher and more sustained activation of ERK. Preliminary data suggest that sustained ERK activity may explain why high thrombin levels do not rescue Panc1 cells form growth factor deprivation-induced cell death. Indeed, in the presence of an ERK inhibitor, high thrombin levels also rescue cells from growth factor deprivation induced cell death. Conclusion: Thrombin protects Panc1 cells in a dose dependent manner from growth factor deprivation-induced cell death and this seems to be dependent on activation of the mTOR pathway.
PO-82 Hypoxia induces Smad2 phosphorylation signaling and increases stem cell-like properties and multi-drug resistance in K562 leukemic cells X.-Y. Cui 1,2 , G. Skretting 1 , Y. Jing 3 , C.F. Myklebust 1 , Y.-F. Liu 2 , P.M. Sandset 1,4 , L. Sun 2 1 Department of Haematology, Oslo University Hospital, Norway; 2 Department of Hematology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China; 3 Department of Physiology, Zhengzhou University Stem Cell Center, Zhengzhou University, Zhengzhou, China; 4 Institute of Clinical Medicine, University of Oslo, Norway Background: Both leukemia and treatment of the disease with chemotherapy can cause hypercoagulability. In general, chemotherapy is an effecient non-targeted therapy for killing the majority of cells in leukemia. However, leukemia cells resistant to such therapy persist in a hypoxic bone marrow niche after chemotherapy, ultimately resulting in patient relapse. Aim: The aim of the present study was to investigate the potential role of hypoxia in maintaining the stem cell-like properties and therapeu-