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Combination therapy using a novel Plk-1 inhibitor and gemcitabine in pancreatic cancer cells
Abstracts / Pancreatology 14 (2014) S1eS129
remodelling and ADM formation was evaluated in vitro using a 3D culture system of pancreatic explants from mice expressing the activating Kras (G12D) mutation. Results: Lack of 5-HT in vivo or inhibition of its uptake 5-HT in vitro resulted in increased cell-cell adhesion. In both cerulein regimens, WT mice developed typical ADM lesions four days after the induction of pancreatitis. Conversely, TPH-1-/- mice were characterized by stronger cell-cell adhesion and showed smaller ADM foci with incomplete acinar cell trans-differentiation. In vitro, inhibition of intracellular 5-HT up-take completely prevented the formation of ADMs in Kras explants. Conclusion: These data indicate that cytoskeletal remodeling mediated by intracellular 5-HT is critical for the formation of ADM lesions. Thus, inhibition of 5-HT uptake may constitute a novel pharmacological intervention to interfere with ADM formation and prevent the spreading of pancreatic pre-malignant lesions.
O-60. Autophagy mediates resistance to gemcitabine treatment through a novel E2F1-p300-VMP1 pathway Maria I. Vaccaro, Alejandro Ropolo, Maria I. Molejon, Cintia Catrinacio, Felipe Renna, Veronica Boggio, Claudio Gonzalez Department of Pathophysiology, Institute for Biochemistry and Molecular Medicine, University of Buenos Aires, CONICET, Argentina Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human malignancies. Gemcitabine is the standard chemotherapeutic treatment for advanced PDAC despite its poor efficacy. Autophagy is an evolutionarily conserved degradation process of cytoplasmic cellular constituents. There is evidence that autophagy has a significant pro-tumorigenic role in established cancers. Aims: Here we characterize a new molecular pathway mediated by VMP1, an autophagy-related protein inducible by activated K-Ras. Materials & methods: Highly resistant pancreatic cancer cells PANC-1, carrying activated K-Ras and BXPC-3 cells, that do not carry K-Ras mutation were used. We evaluate regulation of VMP1 promoter by Luciferase and Chromatin immunoprecipitation assays. Gemcitabine treatment effects on autophagy, cell survival and cell death were determined. Results: Gemcitabine requires VMP1 expression to induce autophagy in highly resistant PANC-1 cells, but not in BxPC-3 cells. We identified the transcription factor E2F1 as an effector of gemcitabine-induced autophagy. E2F1 binds to the VMP1 promoter in PANC-1 cells regulating the expression and promoter activity of VMP1. We also identified the histone acetyltransferase p300 as a modulator of this promoter activity. Our data show that the E2F1-p300 activator/co-activator complex is part of the regulatory pathway controlling the expression and promoter activity of VMP1 triggered by gemcitabine in PANC 1 cells. Finally, downregulation of VMP1 expression and pharmacological modulation of autophagy sensitize PANC-1 cells to apoptosis and diminish clonogenicity under gemcitabine treatment. Conclusion: Together, these data provide evidence of a transcriptional regulation mechanism of autophagy that integrates this cellular process into the complex network of events involved in PDAC chemoresistance.
O-61. Combination therapy using a novel Plk-1 inhibitor and gemcitabine in pancreatic cancer cells Owain Jones, William (Bill) Greenhalf, Chris Halloran, Paula Ghaneh Department of Molecular and Clinical Cancer Medicine, University of Liverpool, United Kingdom Background: Novel chemotherapeutic agents show promise in the treatment of pancreatic cancer, but trial of these therapies is usually dependent on use with existing standard of care treatment.Gemcitabine is
S21
a standard chemotherapeutic drug used in both the adjuvant and advanced settings, it acts primarily by inhibiting DNA replication, but also has actions affecting transcription at all points in the cell cycle. BI6727 is a novel Plk-1 inhibitor that induces mitotic arrest. Both gemcitabine and BI6727 can induce apoptosis. Aims: To identify the optimal sequence-combination therapy for Plk-1 inhibition with gemcitabine Materials & methods: IC50 and isobolar analysis were carried out using the EZ4U assay (Biomedica). Suit-2, Miapaca-2, BXPC-3 and CFPAC were treated with BI6727 and/or gemcitabine with drugs administered sequentially or simultaneously. For FACS, cells were fixed in 70% ethanol prior to propidium iodide staining. Cell lines were genotyped using STR profiling. Results: The IC50 values for Suit-2, Miapaca-2, BXPC-3 and CFPAC were 21nM, 40nM, 28nM and 18nM respectively for gemcitabine and 81nM, 73nM, 93nM and 115nM for BI6727. Treatment with a combination of gemcitabine and BI6727 suggests antagonism between both drugs (AUC >0.7), which became synergistic after pretreatment with BI6727 for 24 hours (AUC <0.2). FACS analysis confirmed G1 accumulation and partial completion of S-phase with gemcitabine, while BI6727 gave G2 accumulation as expected. In combination, much lower S-phase was seen than with gemcitabine alone. Gemcitabine caused increased G2 accumulation following pretreatment with BI6727. Conclusion: Inhibition of Plk-1 combined with gemcitabine requires pretreatment with the Plk-1 inhibitor. This may allow an increase in the toxicity of the G2 effects of gemcitabine.
O-62. Pharmacologic macrophage depletion affects metastasis formation by modulating systemic immune responses in a genetic pancreatic cancer model Heidi Griesmann, Christof Drexel, Nada Milosevic, Bence Sipos, Thomas M. Gress, Patrick Michl University of Marburg, Dept. of Gastroenterology, Germany Background: Tumour-associated macrophages (TAM) play an important role in mediating tumour progression. In pancreatic cancer, infiltrating macrophages have been identified not only in invasive tumours, but also in early preinvasive pancreatic intraepithelial neoplasias and are known to mediate tumour progression. Aims: We aimed to study the impact of pharmacological macrophage depletion by liposomal clodronate in the genetic mouse model of pancreatic cancer. Materials & methods: KPC mice (LSL-KrasG12D/+;LSL-Trp53R172H/+; Pdx-1-Cre) were treated with liposomal clodronate or control liposomes i.p. from week 8 to week 20. Tumour and metastasis formation as well as alterations in local and circulating immune cells and cytokines were analysed. Results: Treatment with liposomal clodronate effectively reduced CD11b-positive macrophages both in the pancreas and other organs such as liver, lung and spleen. Tumour incidence and size was only slightly reduced. However, metastasis formation in the liver and lungs was markedly diminished after macrophage depletion. Reduced macrophage count was associated with significant alterations in circulating growth factors and mediators known to be secreted by macrophages and associated with angiogenesis, most prominently VEGF. Moreover, application of liposomal clodronate led to marked alterations in circulating immune cells, among them reduced regulatory T cells. Conclusion: Pharmacological depletion of macrophages in a genetic mouse model of pancreatic cancer markedly reduced metastasis formation and is associated with modulations in the profile of secreted mediators and regulatory T cells. Pharmacological modulation of infiltrating macrophages represents a promising avenue for antimetastatic therapeutic approaches.
remodelling and ADM formation was evaluated in vitro using a 3D culture system of pancreatic explants from mice expressing the activating Kras (G12D) mutation. Results: Lack of 5-HT in vivo or inhibition of its uptake 5-HT in vitro resulted in increased cell-cell adhesion. In both cerulein regimens, WT mice developed typical ADM lesions four days after the induction of pancreatitis. Conversely, TPH-1-/- mice were characterized by stronger cell-cell adhesion and showed smaller ADM foci with incomplete acinar cell trans-differentiation. In vitro, inhibition of intracellular 5-HT up-take completely prevented the formation of ADMs in Kras explants. Conclusion: These data indicate that cytoskeletal remodeling mediated by intracellular 5-HT is critical for the formation of ADM lesions. Thus, inhibition of 5-HT uptake may constitute a novel pharmacological intervention to interfere with ADM formation and prevent the spreading of pancreatic pre-malignant lesions.
O-60. Autophagy mediates resistance to gemcitabine treatment through a novel E2F1-p300-VMP1 pathway Maria I. Vaccaro, Alejandro Ropolo, Maria I. Molejon, Cintia Catrinacio, Felipe Renna, Veronica Boggio, Claudio Gonzalez Department of Pathophysiology, Institute for Biochemistry and Molecular Medicine, University of Buenos Aires, CONICET, Argentina Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human malignancies. Gemcitabine is the standard chemotherapeutic treatment for advanced PDAC despite its poor efficacy. Autophagy is an evolutionarily conserved degradation process of cytoplasmic cellular constituents. There is evidence that autophagy has a significant pro-tumorigenic role in established cancers. Aims: Here we characterize a new molecular pathway mediated by VMP1, an autophagy-related protein inducible by activated K-Ras. Materials & methods: Highly resistant pancreatic cancer cells PANC-1, carrying activated K-Ras and BXPC-3 cells, that do not carry K-Ras mutation were used. We evaluate regulation of VMP1 promoter by Luciferase and Chromatin immunoprecipitation assays. Gemcitabine treatment effects on autophagy, cell survival and cell death were determined. Results: Gemcitabine requires VMP1 expression to induce autophagy in highly resistant PANC-1 cells, but not in BxPC-3 cells. We identified the transcription factor E2F1 as an effector of gemcitabine-induced autophagy. E2F1 binds to the VMP1 promoter in PANC-1 cells regulating the expression and promoter activity of VMP1. We also identified the histone acetyltransferase p300 as a modulator of this promoter activity. Our data show that the E2F1-p300 activator/co-activator complex is part of the regulatory pathway controlling the expression and promoter activity of VMP1 triggered by gemcitabine in PANC 1 cells. Finally, downregulation of VMP1 expression and pharmacological modulation of autophagy sensitize PANC-1 cells to apoptosis and diminish clonogenicity under gemcitabine treatment. Conclusion: Together, these data provide evidence of a transcriptional regulation mechanism of autophagy that integrates this cellular process into the complex network of events involved in PDAC chemoresistance.
O-61. Combination therapy using a novel Plk-1 inhibitor and gemcitabine in pancreatic cancer cells Owain Jones, William (Bill) Greenhalf, Chris Halloran, Paula Ghaneh Department of Molecular and Clinical Cancer Medicine, University of Liverpool, United Kingdom Background: Novel chemotherapeutic agents show promise in the treatment of pancreatic cancer, but trial of these therapies is usually dependent on use with existing standard of care treatment.Gemcitabine is
S21
a standard chemotherapeutic drug used in both the adjuvant and advanced settings, it acts primarily by inhibiting DNA replication, but also has actions affecting transcription at all points in the cell cycle. BI6727 is a novel Plk-1 inhibitor that induces mitotic arrest. Both gemcitabine and BI6727 can induce apoptosis. Aims: To identify the optimal sequence-combination therapy for Plk-1 inhibition with gemcitabine Materials & methods: IC50 and isobolar analysis were carried out using the EZ4U assay (Biomedica). Suit-2, Miapaca-2, BXPC-3 and CFPAC were treated with BI6727 and/or gemcitabine with drugs administered sequentially or simultaneously. For FACS, cells were fixed in 70% ethanol prior to propidium iodide staining. Cell lines were genotyped using STR profiling. Results: The IC50 values for Suit-2, Miapaca-2, BXPC-3 and CFPAC were 21nM, 40nM, 28nM and 18nM respectively for gemcitabine and 81nM, 73nM, 93nM and 115nM for BI6727. Treatment with a combination of gemcitabine and BI6727 suggests antagonism between both drugs (AUC >0.7), which became synergistic after pretreatment with BI6727 for 24 hours (AUC <0.2). FACS analysis confirmed G1 accumulation and partial completion of S-phase with gemcitabine, while BI6727 gave G2 accumulation as expected. In combination, much lower S-phase was seen than with gemcitabine alone. Gemcitabine caused increased G2 accumulation following pretreatment with BI6727. Conclusion: Inhibition of Plk-1 combined with gemcitabine requires pretreatment with the Plk-1 inhibitor. This may allow an increase in the toxicity of the G2 effects of gemcitabine.
O-62. Pharmacologic macrophage depletion affects metastasis formation by modulating systemic immune responses in a genetic pancreatic cancer model Heidi Griesmann, Christof Drexel, Nada Milosevic, Bence Sipos, Thomas M. Gress, Patrick Michl University of Marburg, Dept. of Gastroenterology, Germany Background: Tumour-associated macrophages (TAM) play an important role in mediating tumour progression. In pancreatic cancer, infiltrating macrophages have been identified not only in invasive tumours, but also in early preinvasive pancreatic intraepithelial neoplasias and are known to mediate tumour progression. Aims: We aimed to study the impact of pharmacological macrophage depletion by liposomal clodronate in the genetic mouse model of pancreatic cancer. Materials & methods: KPC mice (LSL-KrasG12D/+;LSL-Trp53R172H/+; Pdx-1-Cre) were treated with liposomal clodronate or control liposomes i.p. from week 8 to week 20. Tumour and metastasis formation as well as alterations in local and circulating immune cells and cytokines were analysed. Results: Treatment with liposomal clodronate effectively reduced CD11b-positive macrophages both in the pancreas and other organs such as liver, lung and spleen. Tumour incidence and size was only slightly reduced. However, metastasis formation in the liver and lungs was markedly diminished after macrophage depletion. Reduced macrophage count was associated with significant alterations in circulating growth factors and mediators known to be secreted by macrophages and associated with angiogenesis, most prominently VEGF. Moreover, application of liposomal clodronate led to marked alterations in circulating immune cells, among them reduced regulatory T cells. Conclusion: Pharmacological depletion of macrophages in a genetic mouse model of pancreatic cancer markedly reduced metastasis formation and is associated with modulations in the profile of secreted mediators and regulatory T cells. Pharmacological modulation of infiltrating macrophages represents a promising avenue for antimetastatic therapeutic approaches.