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Signal transducer and activator of transcription 5 (STAT5) expression and activation in pancreatic cancer
Abstracts / Pancreatology 13 (2013) e1–e94
of the mouse pancreas via laparotomy. Two weeks post-cancer cell injection, a second laparotomy was performed to inject drug-loaded MPs or saline into the same tail section of the pancreas. Mouse tissue samples were taken to evaluate the local effects of constant drug release on the pancreatic tumors and to determine the extent of drug escape to the spleen and liver. Positive results of these combined studies will justify additional pre-clinical investigation in a transgenic mouse model of pancreatic cancer.
P148. Signal transducer and activator of transcription 5 (STAT5) expression and activation in pancreatic cancer A. Matsushita 1, H. Sumiyoshi 1, Y. Kawano 1, Y. Mizuguchi 1, M. Yoshioka 1, N. Hagiwara 1, T. Matsutani 1, Y. Nakamura 1, S. Matsumoto 1, T. Aimoto 1, T. Ishiwata 2, Z. NaitoE.Uchida 2,1. 1
Department of Surgery, Nippon Medical School, Tokyo, Japan Department of Pathology, Nippon Medical School, Tokyo, Japan Introduction: Signal Transducer and Activator of Transcription 5 (STAT5) belongs to the 7 family members of cytosolic transcription factors, was first characterized in cytokine- and growth factor dependent signal transduction. STAT5 is comprised two homologous isoforms, STAT5a, and STAT5b. STAT5 has been shown to play a critical role in tumor progression in leukemia, breast, prostate cancer and hepatocellular carcinoma. But the expression, activation and biological role of STAT5 in pancreatic cancer are unknown. Methods and results: Using AsPC-1, BxPC3, Capan-1, HPAF-2, MIA PaCa-2, PANC-1, PK-45H and SW1990 human pancreatic cancer cells, RTPCR, Western blot and confocal microscopy were performed. All of pancreatic cancer cells expressed STAT5a and STAT5b mRNAs and proteins. Especially, PANC-1 and Capan-1 were relatively high levels of STAT5a and STAT5b mRNAs and proteins. Confocal microscopy reveals STAT5a and STAT5b proteins were expressed both in cytoplasm and nuclei in PANC-1 cells. In 28 pancreatic cancer tissues, the nuclear expression rates of STAT5a and STAT5b were 39% and 71 %. Clinicopathologically, STAT5b activation significantly correlated with dissected peripancreatic tissue invasion. Conclusion: STAT5 may contribute to tumor progression in pancreatic cancer. 2
P149. Blockade of the CCK receptor inhibits progression of early PanIN lesions to pancreatic cancer in the Pdx1-Cre, LSL/KrasG12D mouse G. Matters 1, T. Cooper 2, E. Gilius 3, C. McGovern 3, J. Liao 4, J.P. Smith 3. 1 Department of Biochemistry & Molecular Biology, The Pennsylvania State University, Hershey, PA, USA 2 Department of Comparative Medicine & Pathology, The Pennsylvania State University, Hershey, PA, USA 3 Department of Medicine, The Pennsylvania State University, Hershey, PA, USA 4 Department of Public Health Sciences, The Pennsylvania State University, Hershey, PA Background: Cholecystokinin (CCK) has been shown to promote growth of human pancreatic cancer as well as carcinogen-induced pancreatic cancer in animal models. Recently CCK-induced pancreatitis has been shown to accelerate PanIN formation in the LSL/KrasG12D mouse model where the credit has been attributed to the inflammatory response. Hypothesis: We hypothesize that endogenous CCK is necessary for PanIN lesion progression to cancer in the LSL/KrasG12D mouse model and that interruption of the ligand with the CCK receptor will prevent advancement in PanIN grade.
e53
Methods: At 3-4 months of age when 100% of mice should have early PanIN lesions, 48 Pdx-Cre/ LSL/KrasG12D transgenic mice were prospectively randomized according to litter date into one of two groups. The first group received regular drinking water and the second group was given drinking water supplemented with the CCK-receptor antagonist proglumide (0.1 mg/ml). Approximately 8 mice from each group were sacrificed at mos 4, 6, and 8. Pancreata were dissected, paraffin-embedded, and stained with H&E. Histologic sections were examined by the veterinarian pathologist blinded to all treatment groups and age. Tissues were scores for the most abundant pancreatic lesion, highest grade of lesion, and inflammatory and fibrosis score according to the grading system by BermanBooty LD. Results: In the control mice, the most frequent lesion increased from PanIN-2 (focal) at mos 4 to PanIN-3 (focal) lesions at mos 8. In contrast, lesions in mice treated with proglumide failed to progress and the most frequent lesion at mos 8 was the PanIN-1b lesion (p¼0.009). Perilobular and interlobular fibrosis was decreased by 54%, and intralobular fibrosis decreased by 32% in mos 8 mice treated with proglumide compared to controls. Pancreatic inflammation was decreased by 25% in the mos 8 mice treated with proglumide compared to controls. Conclusion: The gastrointestinal peptide, CCK, under normal, physiological conditions is an essential factor in the PanIN to pancreatic cancer progression model. Strategies to block the CCK receptor in high risk patient populations may decrease the incidence of cancer development. Supported by NIH R01 CA117926.
P150. Ribonucleotide reductase subunit M2 levels affect resistance to gemcitabine in vitro J. Melling 1, E. Shaw 1, K. Dajani 1, B. Lane 1, A. Bauer 2, J. Hoheisel 2, J. Neoptolemos 1, W. Greenhalf 1, P. Ghaneh 1. 1 NIHR Pancreas Biomedical Research Unit and CR-UK Pancreas Cancer Centre, University of Liverpool, Liverpool, UK 2 Functional Genome Analysis, DKFZ, Heidelberg, Germany Introduction: Gemcitabine is a standard of chemotherapy for pancreatic cancer in the palliative and adjuvant settings but prognosis remains poor as patients may not respond or develop resistance. Aims: To investigate the mechanisms of gemcitabine resistance developed in vitro. Methods: A resistant cell line (SuitGR) was generated by serial culture of the pancreatic cancer cell line Suit-2 in escalating concentrations of gemcitabine (IC50: 357mM vs 3.4nM in parental cells). Total RNA and protein extracted from parental and SuitGR cells treated or untreated with gemcitabine were analysed using the Illumina Human RNA v3Bead and 810 Protein Antibody microarrays. Differential RNA and protein expression was confirmed using qRT-PCR or western blot. Candidate genes were inhibited using siRNA. Results: The microarrays indicated differential expression of 620 genes at RNA level and 44 genes at protein level, but only 4 transcripts and 5 proteins were independent of treatment, suggesting a role in innate resistance. The most significant transcript was SERPINA1 (75% decreased expression in SuitGR, FDR adjusted p value¼0.002) confirmed with qRTPCR, but knockdown of SERPINA1 did not affect resistance. The most significant protein was RRM2 with a 52% decrease in expression in SuitGR (FDR adjusted p value¼<0.001) confirmed by western blot. Knockdown of RRM2 decreased resistance in both parental (3.4nM vs 0.004nM, p¼<0.001) and SuitGR (357mM vs 29.5mM, p¼<0.001) cells. Conclusion: This study has confirmed that RRM2 is a target of gemcitabine and that forced reduction of expression is associated with enhanced drug sensitivity. Reduced RRM2 expression in the relatively gemcitabine resistant SuitGR cells is surprising, indicating a complex interaction with other drug resistance mechanisms, currently being investigated.
of the mouse pancreas via laparotomy. Two weeks post-cancer cell injection, a second laparotomy was performed to inject drug-loaded MPs or saline into the same tail section of the pancreas. Mouse tissue samples were taken to evaluate the local effects of constant drug release on the pancreatic tumors and to determine the extent of drug escape to the spleen and liver. Positive results of these combined studies will justify additional pre-clinical investigation in a transgenic mouse model of pancreatic cancer.
P148. Signal transducer and activator of transcription 5 (STAT5) expression and activation in pancreatic cancer A. Matsushita 1, H. Sumiyoshi 1, Y. Kawano 1, Y. Mizuguchi 1, M. Yoshioka 1, N. Hagiwara 1, T. Matsutani 1, Y. Nakamura 1, S. Matsumoto 1, T. Aimoto 1, T. Ishiwata 2, Z. NaitoE.Uchida 2,1. 1
Department of Surgery, Nippon Medical School, Tokyo, Japan Department of Pathology, Nippon Medical School, Tokyo, Japan Introduction: Signal Transducer and Activator of Transcription 5 (STAT5) belongs to the 7 family members of cytosolic transcription factors, was first characterized in cytokine- and growth factor dependent signal transduction. STAT5 is comprised two homologous isoforms, STAT5a, and STAT5b. STAT5 has been shown to play a critical role in tumor progression in leukemia, breast, prostate cancer and hepatocellular carcinoma. But the expression, activation and biological role of STAT5 in pancreatic cancer are unknown. Methods and results: Using AsPC-1, BxPC3, Capan-1, HPAF-2, MIA PaCa-2, PANC-1, PK-45H and SW1990 human pancreatic cancer cells, RTPCR, Western blot and confocal microscopy were performed. All of pancreatic cancer cells expressed STAT5a and STAT5b mRNAs and proteins. Especially, PANC-1 and Capan-1 were relatively high levels of STAT5a and STAT5b mRNAs and proteins. Confocal microscopy reveals STAT5a and STAT5b proteins were expressed both in cytoplasm and nuclei in PANC-1 cells. In 28 pancreatic cancer tissues, the nuclear expression rates of STAT5a and STAT5b were 39% and 71 %. Clinicopathologically, STAT5b activation significantly correlated with dissected peripancreatic tissue invasion. Conclusion: STAT5 may contribute to tumor progression in pancreatic cancer. 2
P149. Blockade of the CCK receptor inhibits progression of early PanIN lesions to pancreatic cancer in the Pdx1-Cre, LSL/KrasG12D mouse G. Matters 1, T. Cooper 2, E. Gilius 3, C. McGovern 3, J. Liao 4, J.P. Smith 3. 1 Department of Biochemistry & Molecular Biology, The Pennsylvania State University, Hershey, PA, USA 2 Department of Comparative Medicine & Pathology, The Pennsylvania State University, Hershey, PA, USA 3 Department of Medicine, The Pennsylvania State University, Hershey, PA, USA 4 Department of Public Health Sciences, The Pennsylvania State University, Hershey, PA Background: Cholecystokinin (CCK) has been shown to promote growth of human pancreatic cancer as well as carcinogen-induced pancreatic cancer in animal models. Recently CCK-induced pancreatitis has been shown to accelerate PanIN formation in the LSL/KrasG12D mouse model where the credit has been attributed to the inflammatory response. Hypothesis: We hypothesize that endogenous CCK is necessary for PanIN lesion progression to cancer in the LSL/KrasG12D mouse model and that interruption of the ligand with the CCK receptor will prevent advancement in PanIN grade.
e53
Methods: At 3-4 months of age when 100% of mice should have early PanIN lesions, 48 Pdx-Cre/ LSL/KrasG12D transgenic mice were prospectively randomized according to litter date into one of two groups. The first group received regular drinking water and the second group was given drinking water supplemented with the CCK-receptor antagonist proglumide (0.1 mg/ml). Approximately 8 mice from each group were sacrificed at mos 4, 6, and 8. Pancreata were dissected, paraffin-embedded, and stained with H&E. Histologic sections were examined by the veterinarian pathologist blinded to all treatment groups and age. Tissues were scores for the most abundant pancreatic lesion, highest grade of lesion, and inflammatory and fibrosis score according to the grading system by BermanBooty LD. Results: In the control mice, the most frequent lesion increased from PanIN-2 (focal) at mos 4 to PanIN-3 (focal) lesions at mos 8. In contrast, lesions in mice treated with proglumide failed to progress and the most frequent lesion at mos 8 was the PanIN-1b lesion (p¼0.009). Perilobular and interlobular fibrosis was decreased by 54%, and intralobular fibrosis decreased by 32% in mos 8 mice treated with proglumide compared to controls. Pancreatic inflammation was decreased by 25% in the mos 8 mice treated with proglumide compared to controls. Conclusion: The gastrointestinal peptide, CCK, under normal, physiological conditions is an essential factor in the PanIN to pancreatic cancer progression model. Strategies to block the CCK receptor in high risk patient populations may decrease the incidence of cancer development. Supported by NIH R01 CA117926.
P150. Ribonucleotide reductase subunit M2 levels affect resistance to gemcitabine in vitro J. Melling 1, E. Shaw 1, K. Dajani 1, B. Lane 1, A. Bauer 2, J. Hoheisel 2, J. Neoptolemos 1, W. Greenhalf 1, P. Ghaneh 1. 1 NIHR Pancreas Biomedical Research Unit and CR-UK Pancreas Cancer Centre, University of Liverpool, Liverpool, UK 2 Functional Genome Analysis, DKFZ, Heidelberg, Germany Introduction: Gemcitabine is a standard of chemotherapy for pancreatic cancer in the palliative and adjuvant settings but prognosis remains poor as patients may not respond or develop resistance. Aims: To investigate the mechanisms of gemcitabine resistance developed in vitro. Methods: A resistant cell line (SuitGR) was generated by serial culture of the pancreatic cancer cell line Suit-2 in escalating concentrations of gemcitabine (IC50: 357mM vs 3.4nM in parental cells). Total RNA and protein extracted from parental and SuitGR cells treated or untreated with gemcitabine were analysed using the Illumina Human RNA v3Bead and 810 Protein Antibody microarrays. Differential RNA and protein expression was confirmed using qRT-PCR or western blot. Candidate genes were inhibited using siRNA. Results: The microarrays indicated differential expression of 620 genes at RNA level and 44 genes at protein level, but only 4 transcripts and 5 proteins were independent of treatment, suggesting a role in innate resistance. The most significant transcript was SERPINA1 (75% decreased expression in SuitGR, FDR adjusted p value¼0.002) confirmed with qRTPCR, but knockdown of SERPINA1 did not affect resistance. The most significant protein was RRM2 with a 52% decrease in expression in SuitGR (FDR adjusted p value¼<0.001) confirmed by western blot. Knockdown of RRM2 decreased resistance in both parental (3.4nM vs 0.004nM, p¼<0.001) and SuitGR (357mM vs 29.5mM, p¼<0.001) cells. Conclusion: This study has confirmed that RRM2 is a target of gemcitabine and that forced reduction of expression is associated with enhanced drug sensitivity. Reduced RRM2 expression in the relatively gemcitabine resistant SuitGR cells is surprising, indicating a complex interaction with other drug resistance mechanisms, currently being investigated.