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Epidermal growth factor (EGF) chronic stimulation causes Smad4-dependent signaling bypass in PDAC
Abstracts / Pancreatology 14 (2014) S1eS129
Background: Apelin, the endogenous ligand for a G Protein-Coupled Receptor named APJ, acts as a key regulator of tumour neovascularisation. Its gene is upregulated in one third of human tumours and more specifically in pancreatic ductal adenocarcinoma (PDAC). Aims: PDAC is an asymptomatic and multi-steps disease with late prognosis and no effective therapeutics. Thus, finding new biomarkers and therapeutic targets is crucial and apelin signalling could represent a promising candidate. Materials & methods: Apelin and APJ expression is studied by immunohistochemistry realised on human (40 patients) and K-ras mouse (Lox-Stop-Lox-KrasG12D/+/Pdx1-Cre) pancreatic cancer slides. Apelin signalling function was characterised in the human pancreatic cancer cell line MiaPaCa and the regulation of intracellular effectors after apelin stimulation was analysed by western blot. Furthermore, we characterised cellular effects induced by apelin on cancer cells, especially proliferation, migration and glucose uptake. Results: By immunohistochemistry on human tumours and the K-ras mouse model which recapitulates all the stages of the disease, we show that apelin and its receptor are expressed by tumour and endothelial cells since the first steps of carcinogenesis. Characterisation of apelin signalling function in human pancreatic tumour cells reveals that apelin induces activation of key effectors which regulate several cellular effects. Indeed, apelin induces proliferation, migration and glucose uptake, which are crucial properties involved in cancer progression. Conclusion: A better understanding of apelin signalling function in pancreatic carcinogenesis could lead to the identification of a new therapeutic target and could open new perspectives for the treatment of PDAC.
T-068. Epidermal growth factor (EGF) chronic stimulation causes Smad4-dependent signaling bypass in PDAC Stefania Moz a, Dania Bozzato a, Paola Galozzi a, Lucy Fairclough b, Paddy Tighe b, Ian Todd b, Sergio Pedrazzoli c, Daniela Basso a a
Department of Medicine? DIMED, University of Padova, Italy School of Life Sciences Queen?s Medical Centre, University of Nottingham, United Kingdom c Associazione Wirsung-Onlus, Paova, Italy b
Background: EGF and EGFR overexpression is an early, while Smad4 inactivation is a late event in PDAC progression. Acquired resistance to EGFR-targeted therapies might depend on the activation of bypass signaling pathways. Aims: To verify whether a prolunged exposure of PDAC cells to EGF activates bypass signaling and whether this event is Smad4 dependent. Materials & methods: BxPC3 (Smad4 HD) and Smad4-transfected BxPC3 (BxPC3-Smad4+) remained unstimulated (C) or were daily stimulated (S) for three days with EGF (100ng/mL). Then, EGF treatment (100 ng/ mL for 10 minutes) was followed by cell collection for Reverse Phase Protein Array (RPPA) analysis of: MAPK, NF-kB, SRC/JAK/STAT3, PI3K/AKT, inflammasome signaling pathways. RPPA data analysis consisted of calculating the concentration of each sample, after background correction and normalization (signal intensities gained from a image software). Intensities of stimulated cells were referred to controls and variations higher than 30% were considered. Results: In C-BxPC3, EGF activated MAPK/ERK (pERK1/2) only. In CBxPC3-Smad4+, EGF activated SRC/JAK/STAT3 (pc-SRC;pRIP2;pJAK2), MAPK/ERK (pERK1/2;pMEK 1/2) and SAPK/JNK (pc-Jun;pSEK/MKK4), not
S69
p38 MAPK (pP38;TRAF2;pTAK1). In S-BxPC3 and in S-BxPC3-Smad4+, EGF did not activate any MAPK pathway not SRC/JAK/STAT3. In S-BxPC3Smad4+, EGF activated NF-KB (pIkBa;IKKg;pIRAK1;A20) and inhibited the inflammasome (pSTAT4;ACS;Caspase1;MyD88), while in S-BxPC3 EGF inhibited only the inflammasome pathway. The PI3K/AKT pathway was never affected by EGF. RPPA results were confirmed by WB (pP38;pERK1/ 2;pAKT308;pAKT473). Conclusion: EGRF chronic stimulation causes Smad4-dependent signaling bypass from MAPK to NF-KB in Smad4-expressing cells. Smad4 HD renders PDAC cells low responsive to EGF acute or chronic stimulation. These findings might explain the PDAC-low response rate to EGRF-targeted therapies.
T-069. Metabolic rewiring of PDAC: When collagen feeds tumour cells. Orianne Olivares a, Jared Mayers b, Sophie Lac a, Fabienne Guillaumond a, ez ene a, J er emy Nigri a, Ezequiel Marie-Noelle Lavaut a, Patrice Berth Calvo c, Richard Tomasini a, Juan Iovanna a, Matthew Vander Heiden b, Sophie Vasseur a rologie de Marseille (CRCM), Unite Centre de Recherche en Cance et de la Recherche Me dicale, France 1068, Institut National de la Sante b Koch Institute for Integrative Cancer reasearch and Department of Biology, Massachussets institute of Technology,Dana-Farber Can, United States c Molecular Endocrinology and Oncology Research Center, Research Center of Centre Hospitalier Universitaire de Laval, Canada a
Background: PDAC remains a deadly cancer, partly because of its singular architecture, with tumour cells embedded into a dense stroma mainly composed of fibroblasts and collagens. Lowering vascularization, hence oxygen/nutrients supplies within the tumour, it creates a drastic environment. To survive, tumour cells develop several adaptations among which metabolic ones. As a consequence, adapted cells become the most agressive. Aims: Regarding metabolic rewiring, aerobic glycolysis is the most studied. Nevertheless glucose alone cannot answer to the huge tumour energetic/biomass needs and discovering alternative tumoural metabolic substrates is highly important. Materials & methods: We used Pdx1-Cre;KRas G12D;Inka4/arf fl/fl PDAC mice models combined with radioactive metabolic tracings to highlight collagen derived pathways, and confirmed their importance in PDAC survival through FACS, proliferation tests and microscopy. Results: PDAC strongly expresses collagen/proline catabolism enzymes compared with sane pancreata. Using immunohistochemical stainings, we confirmed the presence of high amount of collagens (subtypes I and IV), surrounding tumour cells. We correlated both data to the Ecophagy hypothesis stipulating that collagen degradation may fuel tumour cells with proline (representing about 25% of the molecule) especially under nutrient stress. Albeit interesting, this hypothesis was never clearly showed. We herein observe gradual collagen uptake by tumour cells correlated with the force of nutrient deprivations, highlighting collagen as a potent metabolic substrate. Mimicking its degradation, we confirm proline uptake under glucose depletion, leading to an increase of epithelial tumour cells viability. Tracing experiments demonstrate the lead of proline within mitochondria giving Aspartate/Malate involved in biomass production. Conclusion: Collagen is an abundant source of metabolites for PDAC.
Background: Apelin, the endogenous ligand for a G Protein-Coupled Receptor named APJ, acts as a key regulator of tumour neovascularisation. Its gene is upregulated in one third of human tumours and more specifically in pancreatic ductal adenocarcinoma (PDAC). Aims: PDAC is an asymptomatic and multi-steps disease with late prognosis and no effective therapeutics. Thus, finding new biomarkers and therapeutic targets is crucial and apelin signalling could represent a promising candidate. Materials & methods: Apelin and APJ expression is studied by immunohistochemistry realised on human (40 patients) and K-ras mouse (Lox-Stop-Lox-KrasG12D/+/Pdx1-Cre) pancreatic cancer slides. Apelin signalling function was characterised in the human pancreatic cancer cell line MiaPaCa and the regulation of intracellular effectors after apelin stimulation was analysed by western blot. Furthermore, we characterised cellular effects induced by apelin on cancer cells, especially proliferation, migration and glucose uptake. Results: By immunohistochemistry on human tumours and the K-ras mouse model which recapitulates all the stages of the disease, we show that apelin and its receptor are expressed by tumour and endothelial cells since the first steps of carcinogenesis. Characterisation of apelin signalling function in human pancreatic tumour cells reveals that apelin induces activation of key effectors which regulate several cellular effects. Indeed, apelin induces proliferation, migration and glucose uptake, which are crucial properties involved in cancer progression. Conclusion: A better understanding of apelin signalling function in pancreatic carcinogenesis could lead to the identification of a new therapeutic target and could open new perspectives for the treatment of PDAC.
T-068. Epidermal growth factor (EGF) chronic stimulation causes Smad4-dependent signaling bypass in PDAC Stefania Moz a, Dania Bozzato a, Paola Galozzi a, Lucy Fairclough b, Paddy Tighe b, Ian Todd b, Sergio Pedrazzoli c, Daniela Basso a a
Department of Medicine? DIMED, University of Padova, Italy School of Life Sciences Queen?s Medical Centre, University of Nottingham, United Kingdom c Associazione Wirsung-Onlus, Paova, Italy b
Background: EGF and EGFR overexpression is an early, while Smad4 inactivation is a late event in PDAC progression. Acquired resistance to EGFR-targeted therapies might depend on the activation of bypass signaling pathways. Aims: To verify whether a prolunged exposure of PDAC cells to EGF activates bypass signaling and whether this event is Smad4 dependent. Materials & methods: BxPC3 (Smad4 HD) and Smad4-transfected BxPC3 (BxPC3-Smad4+) remained unstimulated (C) or were daily stimulated (S) for three days with EGF (100ng/mL). Then, EGF treatment (100 ng/ mL for 10 minutes) was followed by cell collection for Reverse Phase Protein Array (RPPA) analysis of: MAPK, NF-kB, SRC/JAK/STAT3, PI3K/AKT, inflammasome signaling pathways. RPPA data analysis consisted of calculating the concentration of each sample, after background correction and normalization (signal intensities gained from a image software). Intensities of stimulated cells were referred to controls and variations higher than 30% were considered. Results: In C-BxPC3, EGF activated MAPK/ERK (pERK1/2) only. In CBxPC3-Smad4+, EGF activated SRC/JAK/STAT3 (pc-SRC;pRIP2;pJAK2), MAPK/ERK (pERK1/2;pMEK 1/2) and SAPK/JNK (pc-Jun;pSEK/MKK4), not
S69
p38 MAPK (pP38;TRAF2;pTAK1). In S-BxPC3 and in S-BxPC3-Smad4+, EGF did not activate any MAPK pathway not SRC/JAK/STAT3. In S-BxPC3Smad4+, EGF activated NF-KB (pIkBa;IKKg;pIRAK1;A20) and inhibited the inflammasome (pSTAT4;ACS;Caspase1;MyD88), while in S-BxPC3 EGF inhibited only the inflammasome pathway. The PI3K/AKT pathway was never affected by EGF. RPPA results were confirmed by WB (pP38;pERK1/ 2;pAKT308;pAKT473). Conclusion: EGRF chronic stimulation causes Smad4-dependent signaling bypass from MAPK to NF-KB in Smad4-expressing cells. Smad4 HD renders PDAC cells low responsive to EGF acute or chronic stimulation. These findings might explain the PDAC-low response rate to EGRF-targeted therapies.
T-069. Metabolic rewiring of PDAC: When collagen feeds tumour cells. Orianne Olivares a, Jared Mayers b, Sophie Lac a, Fabienne Guillaumond a, ez ene a, J er emy Nigri a, Ezequiel Marie-Noelle Lavaut a, Patrice Berth Calvo c, Richard Tomasini a, Juan Iovanna a, Matthew Vander Heiden b, Sophie Vasseur a rologie de Marseille (CRCM), Unite Centre de Recherche en Cance et de la Recherche Me dicale, France 1068, Institut National de la Sante b Koch Institute for Integrative Cancer reasearch and Department of Biology, Massachussets institute of Technology,Dana-Farber Can, United States c Molecular Endocrinology and Oncology Research Center, Research Center of Centre Hospitalier Universitaire de Laval, Canada a
Background: PDAC remains a deadly cancer, partly because of its singular architecture, with tumour cells embedded into a dense stroma mainly composed of fibroblasts and collagens. Lowering vascularization, hence oxygen/nutrients supplies within the tumour, it creates a drastic environment. To survive, tumour cells develop several adaptations among which metabolic ones. As a consequence, adapted cells become the most agressive. Aims: Regarding metabolic rewiring, aerobic glycolysis is the most studied. Nevertheless glucose alone cannot answer to the huge tumour energetic/biomass needs and discovering alternative tumoural metabolic substrates is highly important. Materials & methods: We used Pdx1-Cre;KRas G12D;Inka4/arf fl/fl PDAC mice models combined with radioactive metabolic tracings to highlight collagen derived pathways, and confirmed their importance in PDAC survival through FACS, proliferation tests and microscopy. Results: PDAC strongly expresses collagen/proline catabolism enzymes compared with sane pancreata. Using immunohistochemical stainings, we confirmed the presence of high amount of collagens (subtypes I and IV), surrounding tumour cells. We correlated both data to the Ecophagy hypothesis stipulating that collagen degradation may fuel tumour cells with proline (representing about 25% of the molecule) especially under nutrient stress. Albeit interesting, this hypothesis was never clearly showed. We herein observe gradual collagen uptake by tumour cells correlated with the force of nutrient deprivations, highlighting collagen as a potent metabolic substrate. Mimicking its degradation, we confirm proline uptake under glucose depletion, leading to an increase of epithelial tumour cells viability. Tracing experiments demonstrate the lead of proline within mitochondria giving Aspartate/Malate involved in biomass production. Conclusion: Collagen is an abundant source of metabolites for PDAC.