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Mo1470 Activation of the Apelin(Apelin Receptor Axis in Cholangiocarcinoma Promotes Tumor Growth and Angiogenesis
received high numbers of blood transfusions both in endemic (Pakistan) and non-endemic (UK) areas in the setting of chemotherapy treatment The persistently elevated liver enzymes and HEV RNA raise the concern for chronic HEV, especially give his immunosuppressive status A diagnosis of chronic HEV would make it more likely for the patient to be infected with autochthonous HEV with genotype 3 or 4, via blood transfusions or exposure to infected pigs
Mo1472 Secretion of Vascular Endothelial Growth Factor-C by Cancer-Associated Fibroblasts (CAF) Is Stimulated by Platelet-Derived Growth Factor D (PDGFD) and Promotes Lymphangiogenesis in Cholangiocarcinoma Massimiliano Cadamuro, Marta Vismara, Simone Brivio, Andrea Bovo, Mario Strazzabosco, Luca Fabris Cholangiocarcinoma (CCA) is an aggressive liver epithelial malignancy characterized by strong invasiveness and dismal prognosis. The only potentially curative options are surgical resection and liver transplantation, but both are often precluded by the early metastasisation of CCA to the regional lymph nodes. Metastatic spread is favored by an abundant tumor reactive stroma, mainly composed by cancer-associated fibroblasts (CAF) recruited by PDGFD secreted by CCA cells, developing in conjunction with a rich lymphatic vasculature adjacent to the neoplastic ducts. Mechanisms of lymphangiogenesis are largely unexplored. Therefore, we investigated the possible role of PDGF-D and CAF in this process. Methods/ Results. Human CCA specimens were stained with D2-40 (lymphatic endothelial cell marker, LEC), and aSMA (CAF) alone or in combination with antibodies against VEGF-C or its cognate receptor VEGFR-3 (n=6). In CCA, VEGFR-3 positive LEC laid in close vicinity to VEGF-C expressing CAF. Secretion of VEGF-A, VEGF-C, VEGF-D, Ang-1 and Ang-2 (growth factors regulating lymphangiogenesis), was then evaluated in human primary cultured fibroblasts challenged with PDGF-D (n=3). Upon PDGF-D stimulation, fibroblasts increased the levels of VEGF-C, whilst secretion of VEGF-A and Ang-1 did not change; VEGF-D and Ang2 were not expressed. In fibroblasts, PDGF-D-stimulated VEGF-C secretion was significantly blunted by blocking either PDGFRb (the cognate receptor of PDGF-D) with imatinib mesylate or JNK and ERK/NF-kB signaling with specific inhibitors (SP600125, U0126 and BAY117082, respectively). Recruitment (transwell migration) and vascular assembly (lumen diameter, tube elongation and number of intercellular junctions) of LEC were measured after exposure to conditioned medium (CM) harvested from cultured fibroblasts following PDGFD stimulation (n=6). Fibroblast CM stimulated LEC recruitment and their vascular assembly, to an extent comparable to that induced by VEGF-C alone. CM effects on LEC were inhibited by antagonizing PDGFRb in fibroblasts and by inhibiting both VEGFR-2 and VEGFR-3 in LEC (n=6). In conclusion, in CCA an intense crosstalk involving CCA cells, CAF and LEC, and mediated by PDGF-D and VEGF-C, orchestrates tumoral lymphangiogenesis. Besides recruiting CAF in the tumor reactive stroma, PDGF-D secreted by CCA cells stimulates CAF to release VEGF-C through the JNK and ERK/NF-kB pathways. In turn, VEGF-C promotes tumor lymphangiogenesis acting on VEGFR-2 and VEGFR-3 expressed by LEC. This mechanism identifies several targets (PDGFR b, JNK and ERK/NF-kB, VEGFR-2 and VEGFR-3) amenable of interference to prevent early lymph node metastasisation in CCA.
Mo1469
AASLD Abstracts
Fibroblast Growth Factor Receptor (FGFR) Signaling Activation Culminates in a Mcl-1 Regulated Tumor Survival Pathway in CC Sumera Rizvi, Daisaku Yamada, Petra Hirsova, Steven F. Bronk, Nathan W. Werneburg, Liang Zhang, Eugenia Trushina, Gregory J. Gores Background and Aims: Cholangiocarcinoma (CCA) is a highly lethal biliary tract neoplasm with limited therapeutic options and abysmal overall survival. We have identified an autocrine oncogenic, feed-forward signaling loop between fibroblast growth factor receptor and Hippo signaling pathways in human CCA cells.1 However, the therapeutic consequences of disrupting this cross talk have yet to be interrogated. Methods: KMCH and KMBC human CCA cell lines were used for in vitro studies. Cell death was quantified by biochemical and morphologic criteria and mitochondrial oxidative metabolism by XF24 Seahorse technology. In vivo studies were conducted in CL57BL/6 mice (N=20) which underwent biliary transduction of constitutively active AKT and YAP, a novel model of murine carcinogenesis recently developed by us. From weeks 6-8, mice received either vehicle or the fibroblast growth factor receptor (FGFR) specific inhibitor, BGJ398, via daily oral gavage. All mice were sacrificed at the end of week 8 and tumor burden and biology examined. Results: FGFR inhibition by BGJ398 resulted in cell death of both KMBC and KMCH cells (53% and 36% at 24 hrs, respectively). Profiling Bcl-2 family members by immunoblot analysis revealed specific loss of the survival protein Mcl-1 following BGJ398 treatment. Interestingly, cell death was not accompanied by an increase in caspase activity nor blocked by the caspase inhibitor Q-VD-OPh and appeared to be necrotic rather than apoptotic. Further interrogation of the cell death pathways identified impairment of mitochondrial metabolic oxidation as assessed by oxygen consumption rates (OCR) and cellular ATP levels during BGJ398 treatment; these functional impairments and cell viability were restored with enforced Mcl-1 expression. In our mouse model of CCA, BGJ398 treatment resulted in a significant reduction in tumor burden and increase in tumor cell death as assessed by the TUNEL assay. In Conclusion, inhibition of the FGFR/Hippo autocrine pathway by BGJ398, a pan-FGFR inhibitor, resulted in cell death associated with cellular depletion of Mcl-1; a necrotic form of cell death heretofore not associated with Mcl-1 function. Moreover, in a murine genetic model of CCA, BGJ398 had a significant tumor suppressive effect. Thus, inhibition of FGFR represents a promising therapeutic approach in YAP-driven human CCA.
Mo1473 Autocrine Stress-Induced Phosphoprotein 1 Signaling Promotes Hepatocellular Carcinoma Cell Growth Through Pro-Proliferative and Anti-Apoptotic Pathways Sui Peng, Yanyan Zhang, Tianhong Su, Lixia Xu, Zhirong Zeng, Min-hu Chen
Mo1470 Activation of the Apelin Apelin Receptor Axis in Cholangiocarcinoma Promotes Tumor Growth and Angiogenesis Chad Hall, Laurent Ehrlich, Tori White, April O'Brien, Terry C. Lairmore, Gianfranco Alpini, Shannon Glaser
Background: Stress-induced phosphoprotein 1 (STIP1) is well recognized as an adaptor protein that bridges HSP70 and HSP90 in protein folding. Recent studies reported that STIP1 could also act as a secretory protein to promote malignant cell growth. However, the role of STIP1 in hepatocellular carcinoma (HCC) remains unknown. Methods: STIP1 protein levels were detected in 231 HCC patients by immunohistochemistry and their correlation with prognosis was analyzed. STIP1 protein was measured by Western blot in 28 paired fresh HCC tumor and adjacent samples. ELISA was performed to assess serum STIP1 levels from 25 HCC patients and 25 normal controls. Mechanistically, effects of STIP1 on HCC cell proliferation and apoptosis were examined in vitro and in vivo. Result: STIP1 protein levels were significantly higher in HCC tissues than those in adjacent normal tissues, In HCC patients, STIP1 expression was associated with tumor progression and poor survival, and the serum STIP1 was higher than that in healthy controls. Mechanistically, we demonstrated that STIP1 promoted HCC cell growth through PLC-Erk1/2-dependent pro-proliferative and PI3K-AKT-dependent anti-apoptotic pathway. This STIP1 mediated cell growth was self-sustainable through an autocrine positive feed-back loop, which could be suppressed either by neutralizing extracellular STIP1 or by knocking down intracellular STIP1. In xenograft mice, knockdown of STIP1 significantly reduced tumor growth. Conclusion: We identified STIP1 as a molecular marker and prognostic factor for HCC. Our findings that blocking either intercellular or extracellular STIP1 activity suppressed tumor cell growth provide supports for STIP1 as potential therapeutic target in HCC.
Background: The apelin receptor is a class A G-protein coupled receptor with wide distribution throughout the human body. This receptor, along with its cognate protein ligand apelin, is responsible for a variety of physiological mechanisms including angiogenesis and cardiovascular contractility. The receptor is also involved in benign and malignant pathologies including diabetes, obesity, colon, prostate and breast cancer. Cholangiocarcinoma (CCA) is a rare, but often-fatal malignancy of intra and extrahepatic cholangiocytes. Proliferating cholangiocytes display neuroendocrine phenotypes that secrete and respond to a number of neuropeptides and hormones creating a neuroendocrine compartment in the liver. On this background, we test the hypothesis that the apelinèapelin receptor axis regulates CCA growth by autocrine/paracrine mechanisms. Methods: Several CCA cell lines (CCLP, HuH28, HuCCT-1, SG231, TFK and Mz-ChA-1) and non-malignant cholangiocytes (H69) were used to measure the expression of apelin receptor via immunoblots and/or FACS analysis. Immunohistochemistry (IHC) was also used to measure apelin receptor expression in human CCA tissue arrays. Apelin secretion from CCA and H69 cell lines was measured by ELISA. CCA cell lines were treated with apelin in the presence and absence of apelin receptor antagonist over various time-points. Changes in proliferation were measured using MTS assays and qPCR for Ki-67 and PCNA. Angiogenesis markers (VEGF-A, VEGF-C and its receptors, FLT1, FLT4, Ang1, Ang2 and its receptors Tie1, Tie2) were measured by qPCR. The Muse MAPK Dual Detection kit was used to measure phosphorylation of ERK1/2, a known pathway for cholangiocyte proliferation. Results: There was up-regulation of apelin receptor in CCA cell lines and human CCA tissue arrays compared to non-malignant controls. By ELISA there was enhanced secretion of apelin in CCA lines compared to H69. Treatment of cholangiocarcinoma cells with apelin increased CCA growth, whereas the apelin receptor antagonist significantly decreased basal proliferation and expression of angiogenesis markers. Treatment of CCA cells with apelin increased MAP Kinase activation compared to untreated cells. Conclusion: Apelin secretion and its receptor are up-regulated in cholangiocarcinoma compared to normal controls. Apelin promotes CCA proliferation through activation of the MAP Kinase pathway. Inhibition of the apelin receptor decreases basal proliferation, suggesting an autocrine mechanism of CCA growth. Modulation of the apelin apelin receptor axis may serve as a novel therapeutic strategy to inhibit cholangiocarcinoma tumorigenesis.
AASLD Abstracts
X : 10052$CH02 03-28-16 22:36:37 PDFd : 10052B : e
Mo1474 REC8 Functions as a Tumor Suppressor in Hepatocellular Carcinoma Jing Han, Jia Wang, Xiao-Li Xie, Yun Bai, Xiaoyu Jiang, Ai-Di Li, Qian Ding, Zijin Cui, Jie Yin, MIao-miao Wang, Dong-xuan Zhang, Yi-chao Yang, Lei Chen, Chenguang Ji, Cunkai Wang, Huiqing Jiang Background and Aims: The meiotic cohesin REC8 belongs to the cohesin protein complex, which is essential for correct chromosome disjunction and homologous recombination in the mitotic and meiotic cycle. The role of REC8 in the pathogenesis of hepatocellular carcinoma (HCC) is poorly understood. The objective of this study was to characterize REC8 expression in HCC and to investigate the functional role and the underlying mechanisms of REC8 in HCC. Methods: REC8 expression in human HCC tissues, as compared with the adjacent normal tissues in the same patients, was assessed by immunohistochemistry. The expression level was confirmed by quantitative real-time PCR and western blot. The biological functions of REC8 were determined in vitro by cell viability, colony formation, cell cycle analysis, Annexin V and propidium iodide (PI) double staining cell apoptosis assay and wound healing assay, and in vivo tumorigenicity assay in nude mice. The molecular mechanism of REC8 in hepatocellular carcinoma cell lines was explored by Human Cancer Pathway Array. Results: REC8 expression was down-regulated in human HCC tissues compared to their
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Mo1472 Secretion of Vascular Endothelial Growth Factor-C by Cancer-Associated Fibroblasts (CAF) Is Stimulated by Platelet-Derived Growth Factor D (PDGFD) and Promotes Lymphangiogenesis in Cholangiocarcinoma Massimiliano Cadamuro, Marta Vismara, Simone Brivio, Andrea Bovo, Mario Strazzabosco, Luca Fabris Cholangiocarcinoma (CCA) is an aggressive liver epithelial malignancy characterized by strong invasiveness and dismal prognosis. The only potentially curative options are surgical resection and liver transplantation, but both are often precluded by the early metastasisation of CCA to the regional lymph nodes. Metastatic spread is favored by an abundant tumor reactive stroma, mainly composed by cancer-associated fibroblasts (CAF) recruited by PDGFD secreted by CCA cells, developing in conjunction with a rich lymphatic vasculature adjacent to the neoplastic ducts. Mechanisms of lymphangiogenesis are largely unexplored. Therefore, we investigated the possible role of PDGF-D and CAF in this process. Methods/ Results. Human CCA specimens were stained with D2-40 (lymphatic endothelial cell marker, LEC), and aSMA (CAF) alone or in combination with antibodies against VEGF-C or its cognate receptor VEGFR-3 (n=6). In CCA, VEGFR-3 positive LEC laid in close vicinity to VEGF-C expressing CAF. Secretion of VEGF-A, VEGF-C, VEGF-D, Ang-1 and Ang-2 (growth factors regulating lymphangiogenesis), was then evaluated in human primary cultured fibroblasts challenged with PDGF-D (n=3). Upon PDGF-D stimulation, fibroblasts increased the levels of VEGF-C, whilst secretion of VEGF-A and Ang-1 did not change; VEGF-D and Ang2 were not expressed. In fibroblasts, PDGF-D-stimulated VEGF-C secretion was significantly blunted by blocking either PDGFRb (the cognate receptor of PDGF-D) with imatinib mesylate or JNK and ERK/NF-kB signaling with specific inhibitors (SP600125, U0126 and BAY117082, respectively). Recruitment (transwell migration) and vascular assembly (lumen diameter, tube elongation and number of intercellular junctions) of LEC were measured after exposure to conditioned medium (CM) harvested from cultured fibroblasts following PDGFD stimulation (n=6). Fibroblast CM stimulated LEC recruitment and their vascular assembly, to an extent comparable to that induced by VEGF-C alone. CM effects on LEC were inhibited by antagonizing PDGFRb in fibroblasts and by inhibiting both VEGFR-2 and VEGFR-3 in LEC (n=6). In conclusion, in CCA an intense crosstalk involving CCA cells, CAF and LEC, and mediated by PDGF-D and VEGF-C, orchestrates tumoral lymphangiogenesis. Besides recruiting CAF in the tumor reactive stroma, PDGF-D secreted by CCA cells stimulates CAF to release VEGF-C through the JNK and ERK/NF-kB pathways. In turn, VEGF-C promotes tumor lymphangiogenesis acting on VEGFR-2 and VEGFR-3 expressed by LEC. This mechanism identifies several targets (PDGFR b, JNK and ERK/NF-kB, VEGFR-2 and VEGFR-3) amenable of interference to prevent early lymph node metastasisation in CCA.
Mo1469
AASLD Abstracts
Fibroblast Growth Factor Receptor (FGFR) Signaling Activation Culminates in a Mcl-1 Regulated Tumor Survival Pathway in CC Sumera Rizvi, Daisaku Yamada, Petra Hirsova, Steven F. Bronk, Nathan W. Werneburg, Liang Zhang, Eugenia Trushina, Gregory J. Gores Background and Aims: Cholangiocarcinoma (CCA) is a highly lethal biliary tract neoplasm with limited therapeutic options and abysmal overall survival. We have identified an autocrine oncogenic, feed-forward signaling loop between fibroblast growth factor receptor and Hippo signaling pathways in human CCA cells.1 However, the therapeutic consequences of disrupting this cross talk have yet to be interrogated. Methods: KMCH and KMBC human CCA cell lines were used for in vitro studies. Cell death was quantified by biochemical and morphologic criteria and mitochondrial oxidative metabolism by XF24 Seahorse technology. In vivo studies were conducted in CL57BL/6 mice (N=20) which underwent biliary transduction of constitutively active AKT and YAP, a novel model of murine carcinogenesis recently developed by us. From weeks 6-8, mice received either vehicle or the fibroblast growth factor receptor (FGFR) specific inhibitor, BGJ398, via daily oral gavage. All mice were sacrificed at the end of week 8 and tumor burden and biology examined. Results: FGFR inhibition by BGJ398 resulted in cell death of both KMBC and KMCH cells (53% and 36% at 24 hrs, respectively). Profiling Bcl-2 family members by immunoblot analysis revealed specific loss of the survival protein Mcl-1 following BGJ398 treatment. Interestingly, cell death was not accompanied by an increase in caspase activity nor blocked by the caspase inhibitor Q-VD-OPh and appeared to be necrotic rather than apoptotic. Further interrogation of the cell death pathways identified impairment of mitochondrial metabolic oxidation as assessed by oxygen consumption rates (OCR) and cellular ATP levels during BGJ398 treatment; these functional impairments and cell viability were restored with enforced Mcl-1 expression. In our mouse model of CCA, BGJ398 treatment resulted in a significant reduction in tumor burden and increase in tumor cell death as assessed by the TUNEL assay. In Conclusion, inhibition of the FGFR/Hippo autocrine pathway by BGJ398, a pan-FGFR inhibitor, resulted in cell death associated with cellular depletion of Mcl-1; a necrotic form of cell death heretofore not associated with Mcl-1 function. Moreover, in a murine genetic model of CCA, BGJ398 had a significant tumor suppressive effect. Thus, inhibition of FGFR represents a promising therapeutic approach in YAP-driven human CCA.
Mo1473 Autocrine Stress-Induced Phosphoprotein 1 Signaling Promotes Hepatocellular Carcinoma Cell Growth Through Pro-Proliferative and Anti-Apoptotic Pathways Sui Peng, Yanyan Zhang, Tianhong Su, Lixia Xu, Zhirong Zeng, Min-hu Chen
Mo1470 Activation of the Apelin Apelin Receptor Axis in Cholangiocarcinoma Promotes Tumor Growth and Angiogenesis Chad Hall, Laurent Ehrlich, Tori White, April O'Brien, Terry C. Lairmore, Gianfranco Alpini, Shannon Glaser
Background: Stress-induced phosphoprotein 1 (STIP1) is well recognized as an adaptor protein that bridges HSP70 and HSP90 in protein folding. Recent studies reported that STIP1 could also act as a secretory protein to promote malignant cell growth. However, the role of STIP1 in hepatocellular carcinoma (HCC) remains unknown. Methods: STIP1 protein levels were detected in 231 HCC patients by immunohistochemistry and their correlation with prognosis was analyzed. STIP1 protein was measured by Western blot in 28 paired fresh HCC tumor and adjacent samples. ELISA was performed to assess serum STIP1 levels from 25 HCC patients and 25 normal controls. Mechanistically, effects of STIP1 on HCC cell proliferation and apoptosis were examined in vitro and in vivo. Result: STIP1 protein levels were significantly higher in HCC tissues than those in adjacent normal tissues, In HCC patients, STIP1 expression was associated with tumor progression and poor survival, and the serum STIP1 was higher than that in healthy controls. Mechanistically, we demonstrated that STIP1 promoted HCC cell growth through PLC-Erk1/2-dependent pro-proliferative and PI3K-AKT-dependent anti-apoptotic pathway. This STIP1 mediated cell growth was self-sustainable through an autocrine positive feed-back loop, which could be suppressed either by neutralizing extracellular STIP1 or by knocking down intracellular STIP1. In xenograft mice, knockdown of STIP1 significantly reduced tumor growth. Conclusion: We identified STIP1 as a molecular marker and prognostic factor for HCC. Our findings that blocking either intercellular or extracellular STIP1 activity suppressed tumor cell growth provide supports for STIP1 as potential therapeutic target in HCC.
Background: The apelin receptor is a class A G-protein coupled receptor with wide distribution throughout the human body. This receptor, along with its cognate protein ligand apelin, is responsible for a variety of physiological mechanisms including angiogenesis and cardiovascular contractility. The receptor is also involved in benign and malignant pathologies including diabetes, obesity, colon, prostate and breast cancer. Cholangiocarcinoma (CCA) is a rare, but often-fatal malignancy of intra and extrahepatic cholangiocytes. Proliferating cholangiocytes display neuroendocrine phenotypes that secrete and respond to a number of neuropeptides and hormones creating a neuroendocrine compartment in the liver. On this background, we test the hypothesis that the apelinèapelin receptor axis regulates CCA growth by autocrine/paracrine mechanisms. Methods: Several CCA cell lines (CCLP, HuH28, HuCCT-1, SG231, TFK and Mz-ChA-1) and non-malignant cholangiocytes (H69) were used to measure the expression of apelin receptor via immunoblots and/or FACS analysis. Immunohistochemistry (IHC) was also used to measure apelin receptor expression in human CCA tissue arrays. Apelin secretion from CCA and H69 cell lines was measured by ELISA. CCA cell lines were treated with apelin in the presence and absence of apelin receptor antagonist over various time-points. Changes in proliferation were measured using MTS assays and qPCR for Ki-67 and PCNA. Angiogenesis markers (VEGF-A, VEGF-C and its receptors, FLT1, FLT4, Ang1, Ang2 and its receptors Tie1, Tie2) were measured by qPCR. The Muse MAPK Dual Detection kit was used to measure phosphorylation of ERK1/2, a known pathway for cholangiocyte proliferation. Results: There was up-regulation of apelin receptor in CCA cell lines and human CCA tissue arrays compared to non-malignant controls. By ELISA there was enhanced secretion of apelin in CCA lines compared to H69. Treatment of cholangiocarcinoma cells with apelin increased CCA growth, whereas the apelin receptor antagonist significantly decreased basal proliferation and expression of angiogenesis markers. Treatment of CCA cells with apelin increased MAP Kinase activation compared to untreated cells. Conclusion: Apelin secretion and its receptor are up-regulated in cholangiocarcinoma compared to normal controls. Apelin promotes CCA proliferation through activation of the MAP Kinase pathway. Inhibition of the apelin receptor decreases basal proliferation, suggesting an autocrine mechanism of CCA growth. Modulation of the apelin apelin receptor axis may serve as a novel therapeutic strategy to inhibit cholangiocarcinoma tumorigenesis.
AASLD Abstracts
X : 10052$CH02 03-28-16 22:36:37 PDFd : 10052B : e
Mo1474 REC8 Functions as a Tumor Suppressor in Hepatocellular Carcinoma Jing Han, Jia Wang, Xiao-Li Xie, Yun Bai, Xiaoyu Jiang, Ai-Di Li, Qian Ding, Zijin Cui, Jie Yin, MIao-miao Wang, Dong-xuan Zhang, Yi-chao Yang, Lei Chen, Chenguang Ji, Cunkai Wang, Huiqing Jiang Background and Aims: The meiotic cohesin REC8 belongs to the cohesin protein complex, which is essential for correct chromosome disjunction and homologous recombination in the mitotic and meiotic cycle. The role of REC8 in the pathogenesis of hepatocellular carcinoma (HCC) is poorly understood. The objective of this study was to characterize REC8 expression in HCC and to investigate the functional role and the underlying mechanisms of REC8 in HCC. Methods: REC8 expression in human HCC tissues, as compared with the adjacent normal tissues in the same patients, was assessed by immunohistochemistry. The expression level was confirmed by quantitative real-time PCR and western blot. The biological functions of REC8 were determined in vitro by cell viability, colony formation, cell cycle analysis, Annexin V and propidium iodide (PI) double staining cell apoptosis assay and wound healing assay, and in vivo tumorigenicity assay in nude mice. The molecular mechanism of REC8 in hepatocellular carcinoma cell lines was explored by Human Cancer Pathway Array. Results: REC8 expression was down-regulated in human HCC tissues compared to their
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