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Lymphotoxin promotes acinar cell reprogramming and accelerates pre-neoplastic conversion in Kras induced pancreatic tumorigenesis
Abstracts / Pancreatology 15 (2015) S1eS141
921. Melatonin and its metabolite AFMK is involved in the apoptosis in human pancreatic carcinoma (PANC-1) cells stimulated by gemcitabine Anna Leja-Szpak, Piotr Pierzchalski, Martyna Jastrzebska, Marta Goralska, Jolanta Jaworek Department of Medical Physiology Faculty of Health Sciences School of Medicine Jagiellonian University Krakow Poland, Poland Introduction: Melatonin is a physiological precursor of biologically active metabolites such as N1-acetyl-N2-formyl-5-methoksykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK). We have reported previously that both melatonin and AFMK stimulated expression of HSP27, HSP70 and HSP90a/b in PANC-1 cells and probably this mechanism could be responsible for interruption of intrinsic proapoptotic pathway. However, the precise mechanism standing behind the synergistic action of melatonin, its metabolites, and chemotherapeutic drug (gemcitabine) remains unknown. Aims: Here in, we present the hypothesis that melatonin and its metabolite AFMK could influence the effect of gemcitabine on pancreatic carcinoma (PANC-1) cells and that this phenomenon is depend on the modulation of apoptotic intrinsic pathway. Materials & methods: PANC-1 culture cells have been incubated with its metabolite AFMK (10-8-10-12M) melatonin (10-8-10-12M), or gemcitabine (10-6M), alone or in combination. Cells were harvested, the cytoplasmic proteins were isolated after 24 and 48 hr of incubation and analyzed employing immunoprecipitation and immunoblotting. Results: Both melatonin or its metabolite, administered to PANC-1 cells in combination with gemcitabine inhibits the production of antiapoptotic proteins HSP70 and IAP-2 without affecting significantly the expression of HSP90 and IAP-1 as compared with the results obtained with gemcitabine alone. Furthermore, these changes were accompanied by down-regulation in bcl-2/bax ratio and reduction in procaspases 9 and 3 expression, followed by a significant increase in the formation of both active casapases. Conclusion: We suggest that melatonin or its metabolite AFMK could improved the anti-tumor effect of gemcitabine in PANC-1 cells through the modulation of apoptotic proteins expression.
1183. Lymphotoxin promotes acinar cell reprogramming and accelerates pre-neoplastic conversion in Kras induced pancreatic tumorigenesis Gitta Maria Seleznik 1, *, Theresia Reding 1, *, Sabrina Sonda 1, Eva Diamantis 2, Aurel Perren 2, Anja Zabel 1, Mathias Heikenwalder 3, *, Rolf Graf 1, * 1
Swiss HPB Center, Visceral & Transplantation Surgery, University Hospital Zurich, Switzerland 2 Institute of Pathology, University of Bern, Switzerland 3 €t München (TUM), Institute for Virology, Technische Universita Helmholtz-Centre Munich, Germany Introduction: Pancreatic inflammation is a well-known risk factor for pancreatic ductal adenocarcinoma (PDAC) development in humans. PDAC initiation is linked to activating mutations in KRAS oncogene. Pancreatic acinar cell transdifferentiation results in acinar-to-ductal metaplasia (ADM), which can give rise to pancreatic intraepithelial neoplasia (PanIN), the most common PDAC precursor. Aims: To explore how lymphotoxin induced inflammation promotes ADM and PanIN development. Materials & methods: We established a new genetic model (LTKC) by intercrossing the p48þ/Cre; Krasþ/G12D (KC) model for pancreatic tumorigenesis, to a transgenic mouse developing spontaneous pancreatitis, due to Lymphotoxin (LT) overexpression. Immunohistochemistry and RT-PCR * Corresponding authors.
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were used to obtain an inflammatory signature. In-vitro experiments were performed to investigate the role of LT in acinar transdifferentiation. Results: Lymphotoxin overexpression dramatically accelerated the development of premalignant PanIN lesions in KC animals. 8 week-old LTKC mice already showed extensive ADM and PanIN lesions coinciding with significant upregulation of inflammatory genes and increased GTPbound Kras. These molecular and phenotypic changes were only observed in 16 week-old KC animals. In vitro experiments also confirmed that acinar cells derived from LTKC-animals formed ADMs significantly faster than acini from KC animals. LT overexpression in wt acinar cells was sufficient to initiate spontaneous transdifferentiation. Consequently, in LT mice phosphorylation of EGF Receptor and activation of downstream EGFR signalling (Ras, Stat) were detected. Conclusion: We conclude that Lymphotoxin may contribute to the initiation of spontaneous and pancreatitis-accelerated PDAC precursor formation: By (1) inducing inflammatory environment (2) regulating acinar cell transdifferentiation, or (3) by activating EGFR e leading to accelerated pre-malignant PanIN lesion development.
934. Minnelide inhibits stromal component synthesis improving drug delivery and survival in pancreatic cancer Sulagna Banerjee, Shrey Modi, Olivia McGinn, Vikas Dudeja, Ashok Saluja Univ. of Minnesota, United States Introduction: Pancreatic cancer stromal-microenvironment is the major reason behind the failing of therapy for this disease. The desmoplasmtic stroma is responsible for compression of vasculature in the tumor resulting in impaired drug delivery and poor prognosis in this disease. Our group has evaluated Minnelide, a water-soluble pro-drug of triptolide in pancreatic cancer with very promising results. The current study evaluates the mechanism by which Minnelide depletes stroma while increasing survival in pancreatic tumor. Aims: Evaluating the effect of Minnelide on pancreatic cancer stroma. Materials & methods: Patient tumor xenografts in SCID mice (PDX) and KRASG12DTP53-PDX-Cre pancreatic tumor mice (KPC) were treated with 0.42mg/kg Minnelide. Immunohistochemistry was used to quantitate the staining for stromal components. qPCR was used to determine expressions of genes involved in degradation and synthesis of stroma. Results: Stromal components (Alpha-SMA, HA and collagen) were decreased in both PDX and KPC models following treatment with Minnelide. Similarly expression of HA synthase (HAS) genes (22% of control in PDX and 35% of control in KPC tumors) and their activity (28% of control in PDX and 31% of control in KPC) were decreased in both tumor models in response to Minnelide. No significant decrease was observed in expression or activity of hyalouronidase genes. Further, 4- times more functional vessels were observed in the treated animals, leading to increased drug delivery into the tumor and 38 day longer median survival compared to control. Conclusion: Our study showed that Minnelide, currently under Phase1 clinical trial, depletes the stroma leading to increased functional vasculature and enhanced drug delivery in the tumor.
935. Inhibition of pancreatic cancer by a kinase inhibitor by targeting mutiple signaling pathways Wen-Chun Hung, Hsiu-Mei Chen, Chia-Hua Tsai National Institute of Cancer Research, National Health Research Institutes, Taiwan Introduction: Foretinib is a multiple kinase inhibitor undergoing different phase clinical trials and exhibits potent inhibitory effect on
921. Melatonin and its metabolite AFMK is involved in the apoptosis in human pancreatic carcinoma (PANC-1) cells stimulated by gemcitabine Anna Leja-Szpak, Piotr Pierzchalski, Martyna Jastrzebska, Marta Goralska, Jolanta Jaworek Department of Medical Physiology Faculty of Health Sciences School of Medicine Jagiellonian University Krakow Poland, Poland Introduction: Melatonin is a physiological precursor of biologically active metabolites such as N1-acetyl-N2-formyl-5-methoksykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK). We have reported previously that both melatonin and AFMK stimulated expression of HSP27, HSP70 and HSP90a/b in PANC-1 cells and probably this mechanism could be responsible for interruption of intrinsic proapoptotic pathway. However, the precise mechanism standing behind the synergistic action of melatonin, its metabolites, and chemotherapeutic drug (gemcitabine) remains unknown. Aims: Here in, we present the hypothesis that melatonin and its metabolite AFMK could influence the effect of gemcitabine on pancreatic carcinoma (PANC-1) cells and that this phenomenon is depend on the modulation of apoptotic intrinsic pathway. Materials & methods: PANC-1 culture cells have been incubated with its metabolite AFMK (10-8-10-12M) melatonin (10-8-10-12M), or gemcitabine (10-6M), alone or in combination. Cells were harvested, the cytoplasmic proteins were isolated after 24 and 48 hr of incubation and analyzed employing immunoprecipitation and immunoblotting. Results: Both melatonin or its metabolite, administered to PANC-1 cells in combination with gemcitabine inhibits the production of antiapoptotic proteins HSP70 and IAP-2 without affecting significantly the expression of HSP90 and IAP-1 as compared with the results obtained with gemcitabine alone. Furthermore, these changes were accompanied by down-regulation in bcl-2/bax ratio and reduction in procaspases 9 and 3 expression, followed by a significant increase in the formation of both active casapases. Conclusion: We suggest that melatonin or its metabolite AFMK could improved the anti-tumor effect of gemcitabine in PANC-1 cells through the modulation of apoptotic proteins expression.
1183. Lymphotoxin promotes acinar cell reprogramming and accelerates pre-neoplastic conversion in Kras induced pancreatic tumorigenesis Gitta Maria Seleznik 1, *, Theresia Reding 1, *, Sabrina Sonda 1, Eva Diamantis 2, Aurel Perren 2, Anja Zabel 1, Mathias Heikenwalder 3, *, Rolf Graf 1, * 1
Swiss HPB Center, Visceral & Transplantation Surgery, University Hospital Zurich, Switzerland 2 Institute of Pathology, University of Bern, Switzerland 3 €t München (TUM), Institute for Virology, Technische Universita Helmholtz-Centre Munich, Germany Introduction: Pancreatic inflammation is a well-known risk factor for pancreatic ductal adenocarcinoma (PDAC) development in humans. PDAC initiation is linked to activating mutations in KRAS oncogene. Pancreatic acinar cell transdifferentiation results in acinar-to-ductal metaplasia (ADM), which can give rise to pancreatic intraepithelial neoplasia (PanIN), the most common PDAC precursor. Aims: To explore how lymphotoxin induced inflammation promotes ADM and PanIN development. Materials & methods: We established a new genetic model (LTKC) by intercrossing the p48þ/Cre; Krasþ/G12D (KC) model for pancreatic tumorigenesis, to a transgenic mouse developing spontaneous pancreatitis, due to Lymphotoxin (LT) overexpression. Immunohistochemistry and RT-PCR * Corresponding authors.
S35
were used to obtain an inflammatory signature. In-vitro experiments were performed to investigate the role of LT in acinar transdifferentiation. Results: Lymphotoxin overexpression dramatically accelerated the development of premalignant PanIN lesions in KC animals. 8 week-old LTKC mice already showed extensive ADM and PanIN lesions coinciding with significant upregulation of inflammatory genes and increased GTPbound Kras. These molecular and phenotypic changes were only observed in 16 week-old KC animals. In vitro experiments also confirmed that acinar cells derived from LTKC-animals formed ADMs significantly faster than acini from KC animals. LT overexpression in wt acinar cells was sufficient to initiate spontaneous transdifferentiation. Consequently, in LT mice phosphorylation of EGF Receptor and activation of downstream EGFR signalling (Ras, Stat) were detected. Conclusion: We conclude that Lymphotoxin may contribute to the initiation of spontaneous and pancreatitis-accelerated PDAC precursor formation: By (1) inducing inflammatory environment (2) regulating acinar cell transdifferentiation, or (3) by activating EGFR e leading to accelerated pre-malignant PanIN lesion development.
934. Minnelide inhibits stromal component synthesis improving drug delivery and survival in pancreatic cancer Sulagna Banerjee, Shrey Modi, Olivia McGinn, Vikas Dudeja, Ashok Saluja Univ. of Minnesota, United States Introduction: Pancreatic cancer stromal-microenvironment is the major reason behind the failing of therapy for this disease. The desmoplasmtic stroma is responsible for compression of vasculature in the tumor resulting in impaired drug delivery and poor prognosis in this disease. Our group has evaluated Minnelide, a water-soluble pro-drug of triptolide in pancreatic cancer with very promising results. The current study evaluates the mechanism by which Minnelide depletes stroma while increasing survival in pancreatic tumor. Aims: Evaluating the effect of Minnelide on pancreatic cancer stroma. Materials & methods: Patient tumor xenografts in SCID mice (PDX) and KRASG12DTP53-PDX-Cre pancreatic tumor mice (KPC) were treated with 0.42mg/kg Minnelide. Immunohistochemistry was used to quantitate the staining for stromal components. qPCR was used to determine expressions of genes involved in degradation and synthesis of stroma. Results: Stromal components (Alpha-SMA, HA and collagen) were decreased in both PDX and KPC models following treatment with Minnelide. Similarly expression of HA synthase (HAS) genes (22% of control in PDX and 35% of control in KPC tumors) and their activity (28% of control in PDX and 31% of control in KPC) were decreased in both tumor models in response to Minnelide. No significant decrease was observed in expression or activity of hyalouronidase genes. Further, 4- times more functional vessels were observed in the treated animals, leading to increased drug delivery into the tumor and 38 day longer median survival compared to control. Conclusion: Our study showed that Minnelide, currently under Phase1 clinical trial, depletes the stroma leading to increased functional vasculature and enhanced drug delivery in the tumor.
935. Inhibition of pancreatic cancer by a kinase inhibitor by targeting mutiple signaling pathways Wen-Chun Hung, Hsiu-Mei Chen, Chia-Hua Tsai National Institute of Cancer Research, National Health Research Institutes, Taiwan Introduction: Foretinib is a multiple kinase inhibitor undergoing different phase clinical trials and exhibits potent inhibitory effect on