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Ribonucleotide reductase M2 subunit as novel target for liposarcoma
S86
Surgical Oncology II
death worldwide. This study examines chemopreventative effects of sulindac, a non-selective COX (cyclooxygenase) inhibitor, in a chemical carcinogenesis model. Sulindac was used to avoid potential cardiovascular risks accompanying prolonged COX-2 inhibition in anticipation of translational clinical applications. METHODS: Fifty female Syrian golden hamsters aged 10 weeks underwent initial single subcutaneous injection with the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP), then ethionine/methionine augmentation pressure. Hamsters underwent twice daily orogastric lavage with vehicle (group 1) or 1 of 3 doses of sulindac (50,100, and 150mg/kg/day). Hamster sacrifice was 12 weeks following BOP initiation. RESULTS: Microscopic examination of H&E stained liver revealed no HCC. Significant decreases in mean glutathione-S-transferase positive (GSTp⫹) precancerous lesion number/animal were found in groups 2-4 versus control (p ⬍ 0.0017, p ⬍ 0.015, p ⬍ 0.043). COX-1 and -2, PGE2, and nuclear Ki-67 immunohistochemistry revealed no GSTp⫹ lesion staining differences in groups 2-4 versus control (p ⬎ 0.05). Serum, plasma, and liver tissue PGE2 ELISAs demonstrated no concentration differences in groups 2-4 versus control (p ⬎ 0.05). Sulindac can affect NF-kB signaling in vitro, but no nuclear p65 (NF-kB activity) staining differences were appreciated (p ⬎ 0.05). CONCLUSIONS: We demonstrate significant dose-dependent GSTp⫹ lesion chemoprevention with sulindac which appears independent of COX-1, COX-2, PGE2, and NF-kB activity. The absence of Ki-67 differences suggests sulindac may provide chemoprevention through lowering the apoptotic threshold.
Ribonucleotide reductase M2 subunit as novel target for liposarcoma Rula C Geha MD, Vicotria Wu MD, Penelope DeCarolis BS, Rachael O’Connor BS, JinHong Chen PhD, Gary Schwartz MD, Samuel Singer MD Memorial Sloan Kettering Cancer Center, New York, NY INTRODUCTION: There is a need to develop new targeted therapy for liposarcoma. Our aim is to identify and validate RRM2 as a target for the treatment of liposarcoma.
J Am Coll Surg
sarcoma cells to 3microM of Triapine for 48 hours induced a 2-fold, 1.5-fold and 1.5-fold increase in apoptosis compared to controls in LS141, DDLS and RDD respectively. siRNA technology achieved a 75% knock-down in RRM2 in our liposarcoma cell lines compared to a non-targeted siRNA-control pool. siRNA-RRM2 knockdown in all 3 liposarcoma cell lines showed a 4, 3 and 2.5-fold increase in apoptosis compared to controls in LS141, DDLS and RDD cells, respectively. CONCLUSIONS: Triapine and siRNA-RRM2 induce significant anti-proliferative activity and apoptosis in dedifferentiated liposarcoma cells. These results show the importance of RRM2 maintaining liposarcoma cell viability and the promise of RRM2 targeted agents for the treatment of liposarcoma with upregulated RRM2.
CXCR4 signaling regulates proliferation in pancreatic cancer precursors Ryan M Thomas MD, Andrew M Lowy MD University of Cincinnati, Cincinnati, OH INTRODUCTION: The chemokine receptor CXCR4 has been implicated as a mediator of invasion and metastasis in numerous tumor types including pancreatic cancer. Despite this, little is known about CXCR4 expression and function in pancreatic intraepithelial neoplasia (PanIN). We thus sought to: 1) Characterize CXCR4 expression in PanIN’s initiated by Kras mutation, 2) Investigate Kras/MAPK signaling as a regulator of CXCR4 expression and 3) Determine the effect of CXCR4 signaling on PanIN cell proliferation. METHODS: In this study we utilized a Kras based murine model of human PanIN developed by our group. CXCR4 expression was characterized by Western and immunohistochemistry. PanIN cells were treated with a MEK1/2 inhibitor, U0126, to investigate the role of MAPK signaling on PanIN expression of CXCR4. Assessment of PanIN proliferation was performed using an Alamar Blue assay following exposure to the CXCR4 ligand, CXCL12.
METHODS: Microarrays and real-time PCR measured and validated RRM2 levels in liposarcoma and normal-fat tissue samples. CyQuant assay measured cell viability after treatment of LS141, DDLS, and RDD cell lines with Triapine (an RRM2 inhibitor). Western blots were performed after siRNA-RRM2 transfection. Guava-Nexin measured apoptosis following siRNA-RRM2 and Triapine treatment.
RESULTS: PanIN’s were found to express CXCR4 beginning at PanIN1A. Expression levels increased during the course of PanIN progression. Following CXCL12 treatment of cultured PanIN cells, U0126 was found to inhibit CXCR4 expression in a dose-dependent manner. CXCL12 stimulation of cultured PanIN cells resulted in an 8 fold increase in proliferation at 24 hours following treatment (p⬍0.01).
RESULTS: RRM2 was 18-fold, 38-fold, and 70-fold overexpressed in well-differentiated, dedifferentiated, and pleomorphic-liposarcoma compared to normal-fat. RRM2 levels in 3 liposarcoma cell lines were elevated compared to normal-fat with LS141 having the highest RRM2 levels. The IC50 of Triapine was 1.5microM at 48 hours for dedifferentiated-liposarcoma cell lines. Exposure of lipo-
CONCLUSIONS: CXCR4 is expressed early in the course of pancreatic carcinogenesis. In the setting of ligand stimulation, CXCR4 expression is dependent on MAPK signaling. In cultured PanIN cells, CXCL12 strongly induces cell proliferation suggesting that CXCR4 signaling may play a significant role in pancreatic cancer progression.
Surgical Oncology II
death worldwide. This study examines chemopreventative effects of sulindac, a non-selective COX (cyclooxygenase) inhibitor, in a chemical carcinogenesis model. Sulindac was used to avoid potential cardiovascular risks accompanying prolonged COX-2 inhibition in anticipation of translational clinical applications. METHODS: Fifty female Syrian golden hamsters aged 10 weeks underwent initial single subcutaneous injection with the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP), then ethionine/methionine augmentation pressure. Hamsters underwent twice daily orogastric lavage with vehicle (group 1) or 1 of 3 doses of sulindac (50,100, and 150mg/kg/day). Hamster sacrifice was 12 weeks following BOP initiation. RESULTS: Microscopic examination of H&E stained liver revealed no HCC. Significant decreases in mean glutathione-S-transferase positive (GSTp⫹) precancerous lesion number/animal were found in groups 2-4 versus control (p ⬍ 0.0017, p ⬍ 0.015, p ⬍ 0.043). COX-1 and -2, PGE2, and nuclear Ki-67 immunohistochemistry revealed no GSTp⫹ lesion staining differences in groups 2-4 versus control (p ⬎ 0.05). Serum, plasma, and liver tissue PGE2 ELISAs demonstrated no concentration differences in groups 2-4 versus control (p ⬎ 0.05). Sulindac can affect NF-kB signaling in vitro, but no nuclear p65 (NF-kB activity) staining differences were appreciated (p ⬎ 0.05). CONCLUSIONS: We demonstrate significant dose-dependent GSTp⫹ lesion chemoprevention with sulindac which appears independent of COX-1, COX-2, PGE2, and NF-kB activity. The absence of Ki-67 differences suggests sulindac may provide chemoprevention through lowering the apoptotic threshold.
Ribonucleotide reductase M2 subunit as novel target for liposarcoma Rula C Geha MD, Vicotria Wu MD, Penelope DeCarolis BS, Rachael O’Connor BS, JinHong Chen PhD, Gary Schwartz MD, Samuel Singer MD Memorial Sloan Kettering Cancer Center, New York, NY INTRODUCTION: There is a need to develop new targeted therapy for liposarcoma. Our aim is to identify and validate RRM2 as a target for the treatment of liposarcoma.
J Am Coll Surg
sarcoma cells to 3microM of Triapine for 48 hours induced a 2-fold, 1.5-fold and 1.5-fold increase in apoptosis compared to controls in LS141, DDLS and RDD respectively. siRNA technology achieved a 75% knock-down in RRM2 in our liposarcoma cell lines compared to a non-targeted siRNA-control pool. siRNA-RRM2 knockdown in all 3 liposarcoma cell lines showed a 4, 3 and 2.5-fold increase in apoptosis compared to controls in LS141, DDLS and RDD cells, respectively. CONCLUSIONS: Triapine and siRNA-RRM2 induce significant anti-proliferative activity and apoptosis in dedifferentiated liposarcoma cells. These results show the importance of RRM2 maintaining liposarcoma cell viability and the promise of RRM2 targeted agents for the treatment of liposarcoma with upregulated RRM2.
CXCR4 signaling regulates proliferation in pancreatic cancer precursors Ryan M Thomas MD, Andrew M Lowy MD University of Cincinnati, Cincinnati, OH INTRODUCTION: The chemokine receptor CXCR4 has been implicated as a mediator of invasion and metastasis in numerous tumor types including pancreatic cancer. Despite this, little is known about CXCR4 expression and function in pancreatic intraepithelial neoplasia (PanIN). We thus sought to: 1) Characterize CXCR4 expression in PanIN’s initiated by Kras mutation, 2) Investigate Kras/MAPK signaling as a regulator of CXCR4 expression and 3) Determine the effect of CXCR4 signaling on PanIN cell proliferation. METHODS: In this study we utilized a Kras based murine model of human PanIN developed by our group. CXCR4 expression was characterized by Western and immunohistochemistry. PanIN cells were treated with a MEK1/2 inhibitor, U0126, to investigate the role of MAPK signaling on PanIN expression of CXCR4. Assessment of PanIN proliferation was performed using an Alamar Blue assay following exposure to the CXCR4 ligand, CXCL12.
METHODS: Microarrays and real-time PCR measured and validated RRM2 levels in liposarcoma and normal-fat tissue samples. CyQuant assay measured cell viability after treatment of LS141, DDLS, and RDD cell lines with Triapine (an RRM2 inhibitor). Western blots were performed after siRNA-RRM2 transfection. Guava-Nexin measured apoptosis following siRNA-RRM2 and Triapine treatment.
RESULTS: PanIN’s were found to express CXCR4 beginning at PanIN1A. Expression levels increased during the course of PanIN progression. Following CXCL12 treatment of cultured PanIN cells, U0126 was found to inhibit CXCR4 expression in a dose-dependent manner. CXCL12 stimulation of cultured PanIN cells resulted in an 8 fold increase in proliferation at 24 hours following treatment (p⬍0.01).
RESULTS: RRM2 was 18-fold, 38-fold, and 70-fold overexpressed in well-differentiated, dedifferentiated, and pleomorphic-liposarcoma compared to normal-fat. RRM2 levels in 3 liposarcoma cell lines were elevated compared to normal-fat with LS141 having the highest RRM2 levels. The IC50 of Triapine was 1.5microM at 48 hours for dedifferentiated-liposarcoma cell lines. Exposure of lipo-
CONCLUSIONS: CXCR4 is expressed early in the course of pancreatic carcinogenesis. In the setting of ligand stimulation, CXCR4 expression is dependent on MAPK signaling. In cultured PanIN cells, CXCL12 strongly induces cell proliferation suggesting that CXCR4 signaling may play a significant role in pancreatic cancer progression.