- Email: [email protected]
Mo1948 EGFR Acitvation as a by-Pass Mechanism for the Acquired Resistance of Met Inhibitors in Hepatocellular Carcinoma
advantages in cell motility and anti-apoptosis. Conclusion: HBsAg inhibited the translocation of JTB to the mitochondria and decreased the phosphorylation of p65 through the interaction with JTB, After JTB knockdown, HBsAg exhibited a stronger potential to promote tumor progression. Our data suggested that JTB act as a tumor suppressor gene in regards to HBV infection and its activation might be applied as a therapeutic strategy for in control of HBV related HCC development.
Mo1872 Enhanced Serotonergic Innervation of DRG During Colitis and Its Role in CaMKII-Mediated CREB Activation Chun-Mei Xia, Li-Ya Qiao
Mo1947
The transcription factor CREB (cAMP response element-binding) is implicated to function as a molecular switch underlying neural plasticity, and is activated in the primary sensory neurons in dorsal root ganglia (DRG) during experimental colitis. Activation of CREB is regulated by a list of kinases including Ca2+/calmodulin-dependent protein kinases (CaMK). The neuronal mediators in the sensory pathways contributing to CREB activation in the DRG during colitis are not identified. Recent studies have suggested a role of 5-hydroxytryptamine (5-HT) receptors in mediating sensory activity and visceral hypersensitivity. The AIMS of this study are 1) to examine serotonergic innervation of the DRG during colitis; 2) to examine the expression of metabotropic 5-HT receptor 5-HT4 and its association with phospho (p) -CREB expression in the DRG; and 3) to examine the effects of 5-HT on CREB activation and 5-HT-induced signaling pathways in the DRG. METHODS: Experimental colitis was induced in rats by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS) (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH). Control animals received 50 % EtOH. Rats were killed on day 7 following induction of colitis. The DRG explants were incubated with 5-HT in the presence or absence of various inhibitors. Western blot and immunohistochemistry techniques were used for examination of protein expression. RESULTS: At 7 days of colitis, we have observed robust serotonergic innervation of the L1-L2 DRG where CREB was also activated. The serotonergic fibers contained small and regularly spaced varicosities. The expression level of 5-HT4 was significantly increased in these DRGs, and was co-localized with p-CREB. Application of 5-HT (10 μM) to DRG culture in the presence of Pargyline (1 mM) increased the expression level of p-CREB. 5-HT-elicited CREB activation was blocked by KN-62 (5 μM) and KN-93 (5 μM), the specific inhibitors of CaMKII. During colitis, the phosphorylation level of CaMKII was also increased in DRG neurons expressing p-CREB. CONCLUSION: TNBS colitis-induced CREB activation in primary sensory neurons is likely mediated by 5-HT-induced Ca2+/calmodulin-dependent pathways.
Silencing of Pokemon Attenuates the Proliferation of Hepatocellular Carcinoma HEPG2 Cells In Vitro and In Vivo by Inhibiting the PI3K/AKT Pathway Bayasi Guleng, Chan-Chan Lin, Jian-Lin Ren Members of the POK family of proteins play important roles in embryonic development, differentiation and oncogenesis. Pokemon (POK erythroid myeloid ontogenic factor), which belongs to the POK protein family, is also called LRF, OCZF and FBI-1. As a transcriptional repressor, Pokemon has a critical function in cellular differentiation and oncogenesis. Previous studies have identified an oncogenic role for Pokemon in lymphoma, malignant gliomas and non-small cell lung carcinoma, However, the effects of Pokemon in human hepatocellular carcinoma (HCC) have rarely been studied. Our study demonstrated that Pokemon is overexpressed in human HepG2 cells and that the silencing of Pokemon markedly affected the HepG2 cells. We successfully established HepG2 cell lines, in which Pokemon was stably knocked down. We use the methyl thiazolyl tetrazolium (MTT), bromodeoxyuridine (BrdU) and transwell assays and flow cytometry (FCM) to detect changes in the proliferation, migration and cell cycle progression in HepG2 cells that lacked Pokemon. Using the aforementioned assays, we showed that the silencing of Pokemon inhibited cell growth, proliferation and migration. We also confirmed that Pokemon knock-down inhibited the development of hepatocellular carcinoma In Vivo. We observed that tumor growth was suppressed in nude mice that were transplanted with stable Pokemon knockdown HepG2 cells. In addition, Pokemon may involved in cell cycle progression in HepG2 cells. To elucidate the specific mechanism that was involved, we explored the signaling pathways affected by Pokemon. Our data indicated that silencing of the Pokemon inhibited the PI3K/Akt and c-Raf/MEk/ ERK pathways. These results suggest that Pokemon promotes cell growth, proliferation and migration in hepatocellular carcinoma, which accelerates tumor development via Akt- and ERK- signaling dependent manner.
Mo1873 Mo1948
Catecholaminergic Innervation Regulates Calcium Mobilization and CREB Activation in DRG Neuronal and Glial Cells Li-Ya Qiao, Shanwei Shen, Chun-Mei Xia
EGFR Acitvation as a by-Pass Mechanism for the Acquired Resistance of Met Inhibitors in Hepatocellular Carcinoma C. B. Rountree, Wei Ding
Persistent catecholaminergic innervation of the dorsal root ganglia (DRG) has been demonstrated during visceral hypersensitivity. Release of catecholamine (dopamine, epinephrine and norepinephrine) to the DRG may act on sensory neurons as well as the surrounding glial cells leading to sensory activation. AIMS: this study examined 1) the expression of adrenergic receptors in the DRG neurons and glial cells, and 2) the effects of norepinephrine on the activity of DRG neurons and glial cells. METHODS: Experimental colitis was induced in rats by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS) (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH). Control animals received 50 % EtOH. Rats were killed on day 21 following induction of colitis. The DRG neuronal and glial primary culture was carried out by enzymatic digestion and disassociation of freshly isolated DRG explants, and incubated in DMEM-10. To examine the intracellular calcium ([Ca++]i) level, the culture was pre-loaded with 2 μM fluo-4 acetoxymethyl (AM) for 30 min. Single cell calcium imaging was obtained with a Zeiss AxioImage fluorescence microscope with Apitome z-series imaging and time-lapse photographing. The phosphorylation (activation) level of cAMP response element-binding (CREB) was examined by western blot and immunohistochemistry (IHC). The expression level of calcitonin gene-related peptide (CGRP) was examined by IHC. The presence of adrenergic receptors was examined by polymerase chain reaction (PCR) and IHC. RESULTS: At twenty-one days post colitis induction, the DRG neurons and glial cells exhibited increased expression level of phospho (p)-CREB. The DRG neurons also exhibited increased level of CGRP. Within the DRG, adrenergic receptor β1 was expressed by both neurons and glial cells, and was located in small diameter nociceptors. Application of norepinephrine (25 μM) to DRG explants increased the expression level of CGRP and pCREB in DRG neurons, and p-CREB in surrounding glial cells. Single cell calcium imaging showed that norepinephrine elevated the [Ca++]i level in both DRG neurons and glial cells. CONCLUSION: Catecholaminergic innervation regulates DRG neuronal and glial activities by increasing intracellular calcium levels and CREB activation, implying a role of catecholamine in sensory neuroplasticity.
Hepatocellular carcinoma (HCC) with an active HGF/c-Met signaling pathway has a worse prognosis, and targeting strategies using MET tyrosine kinase inhibitors (TKI) are in early clinical trials for HCC. In non-small cell lung cancer, a by-pass mechanisms for resistance to EGFr TKIs include MET activation. In this report, we utilized human HCC MET+ cell lines to explore a mechanism of EGFr activation during MET TKI therapy, and we examined the effect of combination MET and EGFr TKI therapy on suppressing this by-pass mechanism. Analysis of 449 HCC specimens using published and unpublished transcriptome profiles and immuno-histochemical analysis demonstrates a strong negative correlation between EGFr and MET expression, as co-expression of MET and EGFr was rare. We have previously demonstrated that treatment of human HCC MET+ MHCC97-H xenograft tumors with a MET TKI resulted in stasis of tumor growth In Vivo (You et al. Hepatology. 2011). To understand the mechanism of survival of the MET+ MHCC97-H cells after MET TKI therapy, we developed an MHCC97-H cell line with stable expression of MET shRNA. This MHCC97H METneg line demonstrated the slower growth kinetics and the inhibition of both PI3K and MAPK signaling comparable to MET TKI treated xenografts. Screening against 850 kinases and phosphatases identified EGFr activation as a secondary mechanism of survival in this MHCC97-H METneg line. Gene expression analysis of this MHCC97-H METneg cell line demonstrated a 6 fold increase in TGF-a, a ligand of EGFr. MHCC97-H xenograft tumor analysis demonstrates a 20 fold increase in TGF-a expression after 2 weeks of MET TKI therapy. In Vivo, treatment of MHCC97-H tumors with combination MET and EGFr TKI therapy resulted in complete regression of small tumors compared to partial regression when MET TKI was used as monotherapy. Treatment of large tumors (200 mm3) with combination MET and EGFr TKI therapy resulted in a significant reduction in tumor volume compared to MET TKI monotherapy (Figure 1: 65 mm3 MET/EGFr TKI, 185 mm3 MET TKI, 550600 mm3 for EGFr TKI and untreated groups). To analyze if this combination TKI therapy could be tolerated during liver injury, CCl4 was utilized in mice to induce liver necrosis. Following CCl4, mice reveiving combination EGFr and MET TKI treatment demonstrated significantly reduced liver regeneration at 36 hours (hepatocyte proliferation 35% control vs. 15% MET/EGFr TKI, p<0.05). This initial suppression of hepatocyte proliferation was eventually overcome at 2 weeks, by which time there was no significant difference in liver/ body weight ratio between control vs. MET/EGFr TKI group. Within MET positive HCC, MET TKI therapy results in a by-pass mechanism through up-regulation of TGF-a and activation of EGFr, a by-pass mechanism that can be partially overcome with combination therapy using EGFr and MET TKIs.
Mo1946 HBsAg Inhibits the Translocation of Jtb Into Mitochondria in HEPG2 Cells and Potentially Plays a Role in HCC Progression Bayasi Guleng, Yun-Peng Liu, Jian-Lin Ren Background and Aims: The expression of the jumping translocation breakpoint (JTB) gene is upregulated in malignant liver tissues; however, JTB is associated with unbalanced translocations in many other types of cancer that suppress JTB expression. No comprehensive analysis on its function in human hepatocellular carcinoma (HCC) has been performed to date. We aimed to define the biological consequences for interaction between JTB and HBsAg in HCC cell lines. Methods: We employed the stable transfection to establish small HBsAg expressing HepG2 cell line, and stably silenced the JTB expression using short hairpin RNA in HepG2 cell line. The effects of JTB and small HBsAg In Vitro were determined by assessing cell apoptosis and motility. Results: Silencing of JTB expression promoted cancer cell motility and reduced cell apoptosis. Expression of HBsAg inhibited the translocation of JTB to the mitochondria. Furthermore, silencing of the JTB resulted in an increase in the phosphorylation of p65 in HepG2 cells and HepG2-HBs cells. Whereas HBsAg expression decreased the phosphorylation of p65. The silencing of JTB in HepG2-HBs cells conferred increased
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AGA Abstracts
AGA Abstracts
visceral hyperalgesia. Supported by NIH P50 DK-64539 (YT), 1K01DK088937 (ML) *ML, AM-equal contribution
Mo1951
AGA Abstracts
Interleukin-29 (Interferon-Lambda 1) Inhibits Growth of Hepatocellular Carcinomas in an Orthotope Murine Tumor Model In Vivo Bettina Schwarz, Florian Beigel, Karl W. Jauch, Burkhard Göke, Stephan Brand, Christiane Bruns Objective: Hepatocellular carcinoma (HCC) is a main cause of cancer-related death worldwide and develops in many patients with liver cirrhosis, particularly in patients with chronic viral hepatitis. Treatment options for patients with advanced HCC are limited because of the lack of effective chemotherapeutic agents. Previous In Vitro studies demonstrated that interleukin29 (IL-29, interferon-lambda 1) inhibits hepatitis C virus (HCV) replication in HuH-7 cells and has antiproliferative effects in several tumor cell lines. Aims & Methods: The aim of this study was to investigate potential antiproliferative and antiangiogenic effects of IL-29 In Vitro and in an orthotope HuH-7 tumor model In Vivo. Cell proliferation was analyzed by MTT assays, while cell migration was measured in Boyden chamber experiments. VEGF expression was determined in HuH-7 and HUVEC cells by ELISA. In In Vivo experiments, 6 - 8 week-old male Balb/c nu/nu mice (n=10) were injected orthotopically with 2.5 million HuH-7 cells embedded in Matrigel. From day 7 to 17, the animals were treated with IL-29 i.p. (2μg/kg/d or 4μg/kg/d, respectively) or saline solution. After 2 weeks, the animals were sacrificed and body weight and tumor weight/volume were analysed. Histological analyses of HCC specimens were performed using haematoxylin & eosin-, CD31- and Ki67-staining. Results: Following IL-29 treatment, there was a dose-dependent inhibition of HuH-7 and HUVEC cell proliferation (p<0.003 and p<0.002, respectively) In Vitro. HUVEC cells showed reduced migration rates in Boyden chamber and spheroid assays. VEGF production of HuH7 cells was down-regulated after 24 hours of IL-29 treatment. In In Vivo experiments, there was a reduction of tumor volumes in mice treated with IL-29 (-92.3% for 2μg/kg/d, p<0.02 and -41.3% for 4μg/kg/d, n.s.) compared to non-treated mice. Accordingly, proliferation and tissue staining for microvessel density (MVD) was down-regulated in IL-29 treated mice. Conclusion: IL-29 significantly reduced tumor growth in this murine In Vivo HCC model. Tumor growth inhibition might be mediated by antiproliferative and antiangiogenic effects of IL-29.
Mo1949 Characteristics of Long-Term Survivors With Unresectable Colorectal Liver Metastases Down-Staged by Chemotherapy Followed by Liver Resection Koji Koinuma, Hisanaga Horie, Yasuyuki Miyakura, Masanobu Hyodo, Michitaka Nagase, Hirofumi Fujii, Alan Lefor, Yoshikazu Yasuda Ten to 20 percent of patients with colorectal cancer with unresectable liver metastases meet the criteria for resection after chemotherapy, leading to improved prognosis. In this “Conversion therapy”, however, it remains unclear which patients will have long-term survival. Therefore, we examined the outcomes of such patients to identify characteristics of long-term survivors after “Conversion therapy”. Methods: Multidisciplinary treatment conferences conducted by radiology, digestive surgery and clinical oncology have evaluated patients for possible conversion therapy since 2006. Preoperative chemotherapy included: mFOLFOX6, FOLFIRI or Xelox (+Bevacizumab). Patients were evaluated for resectability every two months. Adjuvant chemotherapy with six months of mFOLFOX6 was administered. Results: Thirty-three patients were enrolled in this study, including 4 patients with a complete remission (CR) (12%) and 17 with a partial response (PR) (52%). Eleven patients underwent hepatic resection (33%; 11/33), including 1 with relapse after CR. Of these 11 patients (7 male, 4 female), age 48-74 years (median 57), 8 had simultaneous lesions, and 3 had metachronous lesions, 4-13 courses of chemotherapy were given before surgery (7). Ten patients underwent an R0 operation. In one patient, tumor nodules were not identified by intra-operative ultrasound, followed by hepatic re-resection after several months. Four of 11 patients are alive without recurrence. Overall survival is 18-55 months (42.5), with 13-40 months after hepatectomy (36), and re-hepatectomy in one case. Lesions disappeared on imaging after chemotherapy in all 4 cases. Pathological CR lesions in the resected specimens were present in 3 cases. Four patients are alive with recurrence. Survival is 4357 months (45.5), along with 6-27 months after relapse (16). Relapse-free survival was more than 12 months after hepatectomy in all 4 cases. Three patients died, with survival of 15-24 months (23), along with 3-8 months after relapse (6). In total, 7 of 11 patients (64%) survived more than 40 months. All the patients originally had tubular adenocarcinoma. Conclusions: Hepatic lesions with a CR (clinically or pathologically) after chemotherapy, along with the pathology of well to moderately differentiated adenocarcinoma, may be favorable factors to predict long-term survival. Long-term relapse-free patients after first hepatectomy (> 12 months) showed long-term survival (> 40 months) even with recurrent cancer. Early recurrence after hepatic resection was associated with a poor prognosis, similar to patients receiving chemotherapy alone.
Mo1952 Invasive Matrix Degradation by Pancreatic Tumor Cells Occurs at Focal Adhesions via Recruitment of the Mt1-MMP Protease by a FAK-P130cas Complex Yu Wang, Mark A. McNiven The metastatic dissemination of pancreatic tumors (PACs) is dependent upon the concomitant degradation of extracellular matrix (ECM) with active cellular migration. It is well established that focal adhesions (FA) play an important role in regulating this migration; however, whether these structures can also contribute to matrix degradation is not clear. The GOAL of this study was to define the mechanisms by which PACs degrade the extracellular environment to facilitate migratory invasion. RESULTS: We have found that multiple pancreatic cancer cell lines display degradation of ECM at FA sites that requires the targeted action of the metalloprotease, MT1-MMP. Importantly, we have found that MT1-MMP targeting to FAs is dependent on an association with a FAK-p130Cas scaffold complex situated at FAs, and is regulated by Src-mediated phosphorylation of Tyr 573 on the cytoplasmic tail of MT1-MMP. Disrupting the FAK-p130Cas-MT1 complex significantly impairs FA-mediated degradation and tumor cell invasion. In CONCLUSION: these findings demonstrate a novel dual function for FAs in PAC cells that include a concomitant degradation of the surrounding matrix while providing substrate adhesion to facilitate invasive migration. This study was funded by NCI CA104125 to MAM.
Mo1950
Mo1953
Chemoresistance in Hepatocellular Carcinoma is Modulated by Interleukin-29 (Interferon-Lambda 1) and Rapamycin Bettina Schwarz, Florian Beigel, Yue Zhao, Karl W. Jauch, Burkhard Göke, Stephan Brand, Christiane Bruns
The Alkaloid and Coffee Constituent Trigonelline Inhibits NRF2 and Sensitizes Pancreatic Cancer Cells for Apoptosis Alexander Arlt, Susanne Krebs, Claudia Geismann, Marie-Luise Kruse, Stefan Schreiber, Susanne Sebens, Heiner Schäfer
Objective: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. Available therapeutics have limitations due to low long-term efficacy and the development of chemoresistance. Therefore, novel therapeutic strategies are warranted to improve survival of patients with HCC. In this study, we investigated In Vitro effects of the novel type-III interferon interleukin-29 (IL-29; interferon-lambda 1) and the mTOR inhibitor rapamycin (sirolimus) as therapeutic strategies for HCC. Aims & Methods: Our aim was to characterize cancer stem cell (CSC) markers on sorafenib-sensitive (Huh7) and sorafenib-resistant (Huh7/ SoR) human HCC cells and to demonstrate the therapeutic effects of IL-29 and rapamycin. To characterize CSC surface markers, FACS and immunofluorescence staining for SP (side population), CD133, CD44, ABCG2 were performed. Cell viability, proliferation and colony formation of sensitive and sorafenib-resistant Huh7 cells under IL-29 and rapamycin treatment were analyzed. Results: We demonstrated a significant dose-dependent inhibition of cell proliferation in both Huh7 and Huh7/SoR cells under IL-29 treatment. The Huh7 SP fraction increased after 72h of therapy with sorafenib at concentration of IC50 and enriched with long term therapy (0.9% at 0h vs. 5.2% at 48h vs. 12.9% at 72h). CD133+ and CD44+ populations increased in Huh7/SoR compared to sensitive Huh7 cells. Rapamycin reduced the SP fraction in sensitive Huh7 and Huh7/SoR cells more effectively than IL-29. Inhibition of colony formation in both sensitive Huh7 and Huh7/SoR cells was more pronounced using rapamycin than IL-29. Immunofluorescence staining showed similar results. Conclusion: There is an obvious effect of IL-29 treatment on sensitive and sorafenib-resistant Huh7 cells. However, rapamycin has a stronger effect on aspects of therapy resistance, concerning the amount of reduced CSC and potential tumor relapse. mTOR inhibition seems to be a promising option as second line therapy in HCC.
Background The transcription factor nuclear factor-E2 related factor-2 (Nrf2) plays an essential role in the development and therapy resistance of many cancer types of cancer, thus pointing to its potential as anti-cancer target but also undermining its established use in chemoprevention. Through the induction of cyto-protective and proteasomal genes, Nrf2 confers apoptosis protection in tumor cells, and inhibiting Nrf2 would be therefore an efficient tool in anti-cancer therapy. Aim The aim of the study was to investigate the feasibility of the alkaloid and coffee constituent trigonelline in the therapy of apoptotic-resistance in pancreatic cancer. Methods and Results In the present study, three pancreatic carcinoma cell lines (Panc1, Colo357, MiaPaca2) as well as the pancreatic duct cell line H6c7 were analysed for the Nrf2 inhibitory effect of trigonelline and its impact on Nrf2 dependent resistance to TRAIL and anti-cancer drug induced apoptosis. Chemoresistant Panc1 and Colo357 cells exhibit high constitutive Nrf2 activity, whereas chemosensitive MiaPaca2 and H6c7 cells display little basal but strong tBHQ-inducible Nrf2 activity and drug resistance. Trigonelline efficiently blocked basal and tBHQ induced Nrf2 activity in all cell lines, exhibiting highest effects at 0.1 μM. Along with Nrf2 inhibition, trigonelline blocked the Nrf2 dependent expression of proteasomal genes such as s5a and alpha5 and reduced proteasome activity in all cell lines tested. These blocking effects were absent if the cells had been treated with Nrf2 siRNA, a condition at which proteasomal gene expression and proteasome activity already decreased. Most notably, the sensitivity of all cell lines to anticancer drug and TRAIL induced apoptosis could be increased by trigonelline, an effect depending not only on Nrf2 but also on proteasomal gene expression. Conclusion We show for the first time that trigonelline is an efficient Nrf2 inhibitor capable of blocking Nrf2 dependent proteasome activity and thereby sensitizing pancreatic cancer cells for apoptotic stimuli. Thus pharmaceutical Nrf2 inhibition might be beneficial in improved anti-cancer therapy.
AGA Abstracts
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Mo1872 Enhanced Serotonergic Innervation of DRG During Colitis and Its Role in CaMKII-Mediated CREB Activation Chun-Mei Xia, Li-Ya Qiao
Mo1947
The transcription factor CREB (cAMP response element-binding) is implicated to function as a molecular switch underlying neural plasticity, and is activated in the primary sensory neurons in dorsal root ganglia (DRG) during experimental colitis. Activation of CREB is regulated by a list of kinases including Ca2+/calmodulin-dependent protein kinases (CaMK). The neuronal mediators in the sensory pathways contributing to CREB activation in the DRG during colitis are not identified. Recent studies have suggested a role of 5-hydroxytryptamine (5-HT) receptors in mediating sensory activity and visceral hypersensitivity. The AIMS of this study are 1) to examine serotonergic innervation of the DRG during colitis; 2) to examine the expression of metabotropic 5-HT receptor 5-HT4 and its association with phospho (p) -CREB expression in the DRG; and 3) to examine the effects of 5-HT on CREB activation and 5-HT-induced signaling pathways in the DRG. METHODS: Experimental colitis was induced in rats by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS) (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH). Control animals received 50 % EtOH. Rats were killed on day 7 following induction of colitis. The DRG explants were incubated with 5-HT in the presence or absence of various inhibitors. Western blot and immunohistochemistry techniques were used for examination of protein expression. RESULTS: At 7 days of colitis, we have observed robust serotonergic innervation of the L1-L2 DRG where CREB was also activated. The serotonergic fibers contained small and regularly spaced varicosities. The expression level of 5-HT4 was significantly increased in these DRGs, and was co-localized with p-CREB. Application of 5-HT (10 μM) to DRG culture in the presence of Pargyline (1 mM) increased the expression level of p-CREB. 5-HT-elicited CREB activation was blocked by KN-62 (5 μM) and KN-93 (5 μM), the specific inhibitors of CaMKII. During colitis, the phosphorylation level of CaMKII was also increased in DRG neurons expressing p-CREB. CONCLUSION: TNBS colitis-induced CREB activation in primary sensory neurons is likely mediated by 5-HT-induced Ca2+/calmodulin-dependent pathways.
Silencing of Pokemon Attenuates the Proliferation of Hepatocellular Carcinoma HEPG2 Cells In Vitro and In Vivo by Inhibiting the PI3K/AKT Pathway Bayasi Guleng, Chan-Chan Lin, Jian-Lin Ren Members of the POK family of proteins play important roles in embryonic development, differentiation and oncogenesis. Pokemon (POK erythroid myeloid ontogenic factor), which belongs to the POK protein family, is also called LRF, OCZF and FBI-1. As a transcriptional repressor, Pokemon has a critical function in cellular differentiation and oncogenesis. Previous studies have identified an oncogenic role for Pokemon in lymphoma, malignant gliomas and non-small cell lung carcinoma, However, the effects of Pokemon in human hepatocellular carcinoma (HCC) have rarely been studied. Our study demonstrated that Pokemon is overexpressed in human HepG2 cells and that the silencing of Pokemon markedly affected the HepG2 cells. We successfully established HepG2 cell lines, in which Pokemon was stably knocked down. We use the methyl thiazolyl tetrazolium (MTT), bromodeoxyuridine (BrdU) and transwell assays and flow cytometry (FCM) to detect changes in the proliferation, migration and cell cycle progression in HepG2 cells that lacked Pokemon. Using the aforementioned assays, we showed that the silencing of Pokemon inhibited cell growth, proliferation and migration. We also confirmed that Pokemon knock-down inhibited the development of hepatocellular carcinoma In Vivo. We observed that tumor growth was suppressed in nude mice that were transplanted with stable Pokemon knockdown HepG2 cells. In addition, Pokemon may involved in cell cycle progression in HepG2 cells. To elucidate the specific mechanism that was involved, we explored the signaling pathways affected by Pokemon. Our data indicated that silencing of the Pokemon inhibited the PI3K/Akt and c-Raf/MEk/ ERK pathways. These results suggest that Pokemon promotes cell growth, proliferation and migration in hepatocellular carcinoma, which accelerates tumor development via Akt- and ERK- signaling dependent manner.
Mo1873 Mo1948
Catecholaminergic Innervation Regulates Calcium Mobilization and CREB Activation in DRG Neuronal and Glial Cells Li-Ya Qiao, Shanwei Shen, Chun-Mei Xia
EGFR Acitvation as a by-Pass Mechanism for the Acquired Resistance of Met Inhibitors in Hepatocellular Carcinoma C. B. Rountree, Wei Ding
Persistent catecholaminergic innervation of the dorsal root ganglia (DRG) has been demonstrated during visceral hypersensitivity. Release of catecholamine (dopamine, epinephrine and norepinephrine) to the DRG may act on sensory neurons as well as the surrounding glial cells leading to sensory activation. AIMS: this study examined 1) the expression of adrenergic receptors in the DRG neurons and glial cells, and 2) the effects of norepinephrine on the activity of DRG neurons and glial cells. METHODS: Experimental colitis was induced in rats by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS) (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH). Control animals received 50 % EtOH. Rats were killed on day 21 following induction of colitis. The DRG neuronal and glial primary culture was carried out by enzymatic digestion and disassociation of freshly isolated DRG explants, and incubated in DMEM-10. To examine the intracellular calcium ([Ca++]i) level, the culture was pre-loaded with 2 μM fluo-4 acetoxymethyl (AM) for 30 min. Single cell calcium imaging was obtained with a Zeiss AxioImage fluorescence microscope with Apitome z-series imaging and time-lapse photographing. The phosphorylation (activation) level of cAMP response element-binding (CREB) was examined by western blot and immunohistochemistry (IHC). The expression level of calcitonin gene-related peptide (CGRP) was examined by IHC. The presence of adrenergic receptors was examined by polymerase chain reaction (PCR) and IHC. RESULTS: At twenty-one days post colitis induction, the DRG neurons and glial cells exhibited increased expression level of phospho (p)-CREB. The DRG neurons also exhibited increased level of CGRP. Within the DRG, adrenergic receptor β1 was expressed by both neurons and glial cells, and was located in small diameter nociceptors. Application of norepinephrine (25 μM) to DRG explants increased the expression level of CGRP and pCREB in DRG neurons, and p-CREB in surrounding glial cells. Single cell calcium imaging showed that norepinephrine elevated the [Ca++]i level in both DRG neurons and glial cells. CONCLUSION: Catecholaminergic innervation regulates DRG neuronal and glial activities by increasing intracellular calcium levels and CREB activation, implying a role of catecholamine in sensory neuroplasticity.
Hepatocellular carcinoma (HCC) with an active HGF/c-Met signaling pathway has a worse prognosis, and targeting strategies using MET tyrosine kinase inhibitors (TKI) are in early clinical trials for HCC. In non-small cell lung cancer, a by-pass mechanisms for resistance to EGFr TKIs include MET activation. In this report, we utilized human HCC MET+ cell lines to explore a mechanism of EGFr activation during MET TKI therapy, and we examined the effect of combination MET and EGFr TKI therapy on suppressing this by-pass mechanism. Analysis of 449 HCC specimens using published and unpublished transcriptome profiles and immuno-histochemical analysis demonstrates a strong negative correlation between EGFr and MET expression, as co-expression of MET and EGFr was rare. We have previously demonstrated that treatment of human HCC MET+ MHCC97-H xenograft tumors with a MET TKI resulted in stasis of tumor growth In Vivo (You et al. Hepatology. 2011). To understand the mechanism of survival of the MET+ MHCC97-H cells after MET TKI therapy, we developed an MHCC97-H cell line with stable expression of MET shRNA. This MHCC97H METneg line demonstrated the slower growth kinetics and the inhibition of both PI3K and MAPK signaling comparable to MET TKI treated xenografts. Screening against 850 kinases and phosphatases identified EGFr activation as a secondary mechanism of survival in this MHCC97-H METneg line. Gene expression analysis of this MHCC97-H METneg cell line demonstrated a 6 fold increase in TGF-a, a ligand of EGFr. MHCC97-H xenograft tumor analysis demonstrates a 20 fold increase in TGF-a expression after 2 weeks of MET TKI therapy. In Vivo, treatment of MHCC97-H tumors with combination MET and EGFr TKI therapy resulted in complete regression of small tumors compared to partial regression when MET TKI was used as monotherapy. Treatment of large tumors (200 mm3) with combination MET and EGFr TKI therapy resulted in a significant reduction in tumor volume compared to MET TKI monotherapy (Figure 1: 65 mm3 MET/EGFr TKI, 185 mm3 MET TKI, 550600 mm3 for EGFr TKI and untreated groups). To analyze if this combination TKI therapy could be tolerated during liver injury, CCl4 was utilized in mice to induce liver necrosis. Following CCl4, mice reveiving combination EGFr and MET TKI treatment demonstrated significantly reduced liver regeneration at 36 hours (hepatocyte proliferation 35% control vs. 15% MET/EGFr TKI, p<0.05). This initial suppression of hepatocyte proliferation was eventually overcome at 2 weeks, by which time there was no significant difference in liver/ body weight ratio between control vs. MET/EGFr TKI group. Within MET positive HCC, MET TKI therapy results in a by-pass mechanism through up-regulation of TGF-a and activation of EGFr, a by-pass mechanism that can be partially overcome with combination therapy using EGFr and MET TKIs.
Mo1946 HBsAg Inhibits the Translocation of Jtb Into Mitochondria in HEPG2 Cells and Potentially Plays a Role in HCC Progression Bayasi Guleng, Yun-Peng Liu, Jian-Lin Ren Background and Aims: The expression of the jumping translocation breakpoint (JTB) gene is upregulated in malignant liver tissues; however, JTB is associated with unbalanced translocations in many other types of cancer that suppress JTB expression. No comprehensive analysis on its function in human hepatocellular carcinoma (HCC) has been performed to date. We aimed to define the biological consequences for interaction between JTB and HBsAg in HCC cell lines. Methods: We employed the stable transfection to establish small HBsAg expressing HepG2 cell line, and stably silenced the JTB expression using short hairpin RNA in HepG2 cell line. The effects of JTB and small HBsAg In Vitro were determined by assessing cell apoptosis and motility. Results: Silencing of JTB expression promoted cancer cell motility and reduced cell apoptosis. Expression of HBsAg inhibited the translocation of JTB to the mitochondria. Furthermore, silencing of the JTB resulted in an increase in the phosphorylation of p65 in HepG2 cells and HepG2-HBs cells. Whereas HBsAg expression decreased the phosphorylation of p65. The silencing of JTB in HepG2-HBs cells conferred increased
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AGA Abstracts
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visceral hyperalgesia. Supported by NIH P50 DK-64539 (YT), 1K01DK088937 (ML) *ML, AM-equal contribution
Mo1951
AGA Abstracts
Interleukin-29 (Interferon-Lambda 1) Inhibits Growth of Hepatocellular Carcinomas in an Orthotope Murine Tumor Model In Vivo Bettina Schwarz, Florian Beigel, Karl W. Jauch, Burkhard Göke, Stephan Brand, Christiane Bruns Objective: Hepatocellular carcinoma (HCC) is a main cause of cancer-related death worldwide and develops in many patients with liver cirrhosis, particularly in patients with chronic viral hepatitis. Treatment options for patients with advanced HCC are limited because of the lack of effective chemotherapeutic agents. Previous In Vitro studies demonstrated that interleukin29 (IL-29, interferon-lambda 1) inhibits hepatitis C virus (HCV) replication in HuH-7 cells and has antiproliferative effects in several tumor cell lines. Aims & Methods: The aim of this study was to investigate potential antiproliferative and antiangiogenic effects of IL-29 In Vitro and in an orthotope HuH-7 tumor model In Vivo. Cell proliferation was analyzed by MTT assays, while cell migration was measured in Boyden chamber experiments. VEGF expression was determined in HuH-7 and HUVEC cells by ELISA. In In Vivo experiments, 6 - 8 week-old male Balb/c nu/nu mice (n=10) were injected orthotopically with 2.5 million HuH-7 cells embedded in Matrigel. From day 7 to 17, the animals were treated with IL-29 i.p. (2μg/kg/d or 4μg/kg/d, respectively) or saline solution. After 2 weeks, the animals were sacrificed and body weight and tumor weight/volume were analysed. Histological analyses of HCC specimens were performed using haematoxylin & eosin-, CD31- and Ki67-staining. Results: Following IL-29 treatment, there was a dose-dependent inhibition of HuH-7 and HUVEC cell proliferation (p<0.003 and p<0.002, respectively) In Vitro. HUVEC cells showed reduced migration rates in Boyden chamber and spheroid assays. VEGF production of HuH7 cells was down-regulated after 24 hours of IL-29 treatment. In In Vivo experiments, there was a reduction of tumor volumes in mice treated with IL-29 (-92.3% for 2μg/kg/d, p<0.02 and -41.3% for 4μg/kg/d, n.s.) compared to non-treated mice. Accordingly, proliferation and tissue staining for microvessel density (MVD) was down-regulated in IL-29 treated mice. Conclusion: IL-29 significantly reduced tumor growth in this murine In Vivo HCC model. Tumor growth inhibition might be mediated by antiproliferative and antiangiogenic effects of IL-29.
Mo1949 Characteristics of Long-Term Survivors With Unresectable Colorectal Liver Metastases Down-Staged by Chemotherapy Followed by Liver Resection Koji Koinuma, Hisanaga Horie, Yasuyuki Miyakura, Masanobu Hyodo, Michitaka Nagase, Hirofumi Fujii, Alan Lefor, Yoshikazu Yasuda Ten to 20 percent of patients with colorectal cancer with unresectable liver metastases meet the criteria for resection after chemotherapy, leading to improved prognosis. In this “Conversion therapy”, however, it remains unclear which patients will have long-term survival. Therefore, we examined the outcomes of such patients to identify characteristics of long-term survivors after “Conversion therapy”. Methods: Multidisciplinary treatment conferences conducted by radiology, digestive surgery and clinical oncology have evaluated patients for possible conversion therapy since 2006. Preoperative chemotherapy included: mFOLFOX6, FOLFIRI or Xelox (+Bevacizumab). Patients were evaluated for resectability every two months. Adjuvant chemotherapy with six months of mFOLFOX6 was administered. Results: Thirty-three patients were enrolled in this study, including 4 patients with a complete remission (CR) (12%) and 17 with a partial response (PR) (52%). Eleven patients underwent hepatic resection (33%; 11/33), including 1 with relapse after CR. Of these 11 patients (7 male, 4 female), age 48-74 years (median 57), 8 had simultaneous lesions, and 3 had metachronous lesions, 4-13 courses of chemotherapy were given before surgery (7). Ten patients underwent an R0 operation. In one patient, tumor nodules were not identified by intra-operative ultrasound, followed by hepatic re-resection after several months. Four of 11 patients are alive without recurrence. Overall survival is 18-55 months (42.5), with 13-40 months after hepatectomy (36), and re-hepatectomy in one case. Lesions disappeared on imaging after chemotherapy in all 4 cases. Pathological CR lesions in the resected specimens were present in 3 cases. Four patients are alive with recurrence. Survival is 4357 months (45.5), along with 6-27 months after relapse (16). Relapse-free survival was more than 12 months after hepatectomy in all 4 cases. Three patients died, with survival of 15-24 months (23), along with 3-8 months after relapse (6). In total, 7 of 11 patients (64%) survived more than 40 months. All the patients originally had tubular adenocarcinoma. Conclusions: Hepatic lesions with a CR (clinically or pathologically) after chemotherapy, along with the pathology of well to moderately differentiated adenocarcinoma, may be favorable factors to predict long-term survival. Long-term relapse-free patients after first hepatectomy (> 12 months) showed long-term survival (> 40 months) even with recurrent cancer. Early recurrence after hepatic resection was associated with a poor prognosis, similar to patients receiving chemotherapy alone.
Mo1952 Invasive Matrix Degradation by Pancreatic Tumor Cells Occurs at Focal Adhesions via Recruitment of the Mt1-MMP Protease by a FAK-P130cas Complex Yu Wang, Mark A. McNiven The metastatic dissemination of pancreatic tumors (PACs) is dependent upon the concomitant degradation of extracellular matrix (ECM) with active cellular migration. It is well established that focal adhesions (FA) play an important role in regulating this migration; however, whether these structures can also contribute to matrix degradation is not clear. The GOAL of this study was to define the mechanisms by which PACs degrade the extracellular environment to facilitate migratory invasion. RESULTS: We have found that multiple pancreatic cancer cell lines display degradation of ECM at FA sites that requires the targeted action of the metalloprotease, MT1-MMP. Importantly, we have found that MT1-MMP targeting to FAs is dependent on an association with a FAK-p130Cas scaffold complex situated at FAs, and is regulated by Src-mediated phosphorylation of Tyr 573 on the cytoplasmic tail of MT1-MMP. Disrupting the FAK-p130Cas-MT1 complex significantly impairs FA-mediated degradation and tumor cell invasion. In CONCLUSION: these findings demonstrate a novel dual function for FAs in PAC cells that include a concomitant degradation of the surrounding matrix while providing substrate adhesion to facilitate invasive migration. This study was funded by NCI CA104125 to MAM.
Mo1950
Mo1953
Chemoresistance in Hepatocellular Carcinoma is Modulated by Interleukin-29 (Interferon-Lambda 1) and Rapamycin Bettina Schwarz, Florian Beigel, Yue Zhao, Karl W. Jauch, Burkhard Göke, Stephan Brand, Christiane Bruns
The Alkaloid and Coffee Constituent Trigonelline Inhibits NRF2 and Sensitizes Pancreatic Cancer Cells for Apoptosis Alexander Arlt, Susanne Krebs, Claudia Geismann, Marie-Luise Kruse, Stefan Schreiber, Susanne Sebens, Heiner Schäfer
Objective: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. Available therapeutics have limitations due to low long-term efficacy and the development of chemoresistance. Therefore, novel therapeutic strategies are warranted to improve survival of patients with HCC. In this study, we investigated In Vitro effects of the novel type-III interferon interleukin-29 (IL-29; interferon-lambda 1) and the mTOR inhibitor rapamycin (sirolimus) as therapeutic strategies for HCC. Aims & Methods: Our aim was to characterize cancer stem cell (CSC) markers on sorafenib-sensitive (Huh7) and sorafenib-resistant (Huh7/ SoR) human HCC cells and to demonstrate the therapeutic effects of IL-29 and rapamycin. To characterize CSC surface markers, FACS and immunofluorescence staining for SP (side population), CD133, CD44, ABCG2 were performed. Cell viability, proliferation and colony formation of sensitive and sorafenib-resistant Huh7 cells under IL-29 and rapamycin treatment were analyzed. Results: We demonstrated a significant dose-dependent inhibition of cell proliferation in both Huh7 and Huh7/SoR cells under IL-29 treatment. The Huh7 SP fraction increased after 72h of therapy with sorafenib at concentration of IC50 and enriched with long term therapy (0.9% at 0h vs. 5.2% at 48h vs. 12.9% at 72h). CD133+ and CD44+ populations increased in Huh7/SoR compared to sensitive Huh7 cells. Rapamycin reduced the SP fraction in sensitive Huh7 and Huh7/SoR cells more effectively than IL-29. Inhibition of colony formation in both sensitive Huh7 and Huh7/SoR cells was more pronounced using rapamycin than IL-29. Immunofluorescence staining showed similar results. Conclusion: There is an obvious effect of IL-29 treatment on sensitive and sorafenib-resistant Huh7 cells. However, rapamycin has a stronger effect on aspects of therapy resistance, concerning the amount of reduced CSC and potential tumor relapse. mTOR inhibition seems to be a promising option as second line therapy in HCC.
Background The transcription factor nuclear factor-E2 related factor-2 (Nrf2) plays an essential role in the development and therapy resistance of many cancer types of cancer, thus pointing to its potential as anti-cancer target but also undermining its established use in chemoprevention. Through the induction of cyto-protective and proteasomal genes, Nrf2 confers apoptosis protection in tumor cells, and inhibiting Nrf2 would be therefore an efficient tool in anti-cancer therapy. Aim The aim of the study was to investigate the feasibility of the alkaloid and coffee constituent trigonelline in the therapy of apoptotic-resistance in pancreatic cancer. Methods and Results In the present study, three pancreatic carcinoma cell lines (Panc1, Colo357, MiaPaca2) as well as the pancreatic duct cell line H6c7 were analysed for the Nrf2 inhibitory effect of trigonelline and its impact on Nrf2 dependent resistance to TRAIL and anti-cancer drug induced apoptosis. Chemoresistant Panc1 and Colo357 cells exhibit high constitutive Nrf2 activity, whereas chemosensitive MiaPaca2 and H6c7 cells display little basal but strong tBHQ-inducible Nrf2 activity and drug resistance. Trigonelline efficiently blocked basal and tBHQ induced Nrf2 activity in all cell lines, exhibiting highest effects at 0.1 μM. Along with Nrf2 inhibition, trigonelline blocked the Nrf2 dependent expression of proteasomal genes such as s5a and alpha5 and reduced proteasome activity in all cell lines tested. These blocking effects were absent if the cells had been treated with Nrf2 siRNA, a condition at which proteasomal gene expression and proteasome activity already decreased. Most notably, the sensitivity of all cell lines to anticancer drug and TRAIL induced apoptosis could be increased by trigonelline, an effect depending not only on Nrf2 but also on proteasomal gene expression. Conclusion We show for the first time that trigonelline is an efficient Nrf2 inhibitor capable of blocking Nrf2 dependent proteasome activity and thereby sensitizing pancreatic cancer cells for apoptotic stimuli. Thus pharmaceutical Nrf2 inhibition might be beneficial in improved anti-cancer therapy.
AGA Abstracts
S-706