- Email: [email protected]
Epigenetic Characterization of RAS Association Domain-Containing Protein 10 as a Functional Tumor Suppressor in Gastric Cancer
AGA Abstracts
disease. TLR2-/-MyD88+/+MDR1A-/- macrophages hyperresponded to LPS stimulation with increased production of IL-1β protein which was not evident in TLR2+/+MyD88+/+MDR1A-/macrophages. Conclusions: Exacerbation of UC-like pancolitis in TLR2/MDR1A double deficiency requires IL-1R/MyD88 signalling. Aberrant LPS-induced IL-1β signalling via IL1R/MyD88 may represent a critical inducer of severe pathology and a novel therapeutic target in a subgroup of UC patients.
PTK6 modulates colon cancer cell sensitivity to chemotherapeutic agents. METHODS: Isogenic HCT116 p53+/+ and p53-/- human colon cancer cell lines containing empty vector or expressing a scrambled shRNA or two different shRNAs that target PTK6 were treated with doxorubicin and 5-fluorouracil, drugs used in cancer chemotherapy. The cell lines were also subjected to 20 Gy of gamma-irradiation and harvested at 0, 3, 6, 24 and 48 hours. Protein lysates were prepared and analyzed by immunoblotting. Cells were incubated with PE Annexin V and 7-Amino-Actinomycin and apoptosis was analyzed by flow cytometry. RNA was prepared and expression of PTK6 and the p53 target gene p21 was examined by qRT-PCR. RESULTS: Stable knockdown of PTK6 in HCT116 p53+/+ and p53-/- cells using two different shRNAs led to increased apoptosis in both HCT116 p53+/+ and HCT116 p53-/- cells with a more dramatic increase in HCT116 p53-/- cells following treatment with doxorubicin, 5-fluorouracil, and radiation. Highest levels of cleaved caspase-3 and apoptosis were detected in PTK6 negative p53-/- cells using immunoblotting and flow cytometry. Rapid accumulation of p53 and activation of the p53 target gene encoding p21 was detected in wild type HCT116 cells following all treatments. Induction of PTK6 was observed by 24 hours after irradiation, with highest levels at 48 hours post irradiation. While PTK6 was induced in both HCT116 p53+/+ and p53-/- cell lines following irradiation, higher levels of PTK6 protein and mRNA were detected in p53+/+ HCT116 cells suggesting a p53dependent component in PTK6 induction. However unlike the p21 gene that is induced by 3 hours post irradiation, the PTK6 gene does not appear to be a direct target of p53. Robust induction of PTK6 was not evident after treatment of HCT116 cells with either doxorubicin or 5-fluorouracil. CONCLUSIONS: PTK6 expression is positively regulated by p53-dependent and independent mechanisms after DNA damage. Knockdown of PTK6 expression promotes apoptosis of HCT116 cells and strongly enhances the response of p53-/colon tumor cells to irradiation, doxorubicin and 5-fluorouracil. Kinase inhibitors targeting PTK6 may enhance sensitivity of colon cancer cells to chemotherapeutic agents.
868 Genome-Wide Binding Analysis of Carboxyl-Terminal Truncated HBx Reveals Direct Repression of a Negative Growth Regulator, GAS2, in Hepatocellular Carcinoma Ranxu Zhu, Alfred S. Cheng, Suki S. Lau, Yangchao Chen, Tin L Lee, Vincent W. Wong, Joseph J. Sung, Henry Lik-Yuen Chan Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) and the X protein of HBV (HBx) is highly implicated in hepatocarcinogenesis. Recently HBx has been shown to act as coregulator of cancer-related genes by interacting with DNAbound transcription factor (TF). The carboxyl-terminal truncated forms of HBx (tHBx), rather than the full-length counterpart, have been shown to frequently express in HCC tissues and possess oncogenic abilities In Vitro and In Vivo. tHBxΔ35 (deletion of 35 carboxylamino acids) is one of the truncated oncoproteins naturally generated by cytidine deaminases in HCC tissues. However, how tHBxΔ35 exerts oncogenic effects is poorly understood. Here we used genome-wide location analysis and functional studies to elucidate the role of direct tHBxΔ35 target genes in the development of HCC. tHBxΔ35 was cloned from the serum of an HCC patient infected with HBV genotype C. Ectopic expression of tHBxΔ35 in 2 immortal human liver cell lines (MIHA and LO2) significantly increased cell proliferation compared with vector-control cells as shown by MTS and colony formation assays (p<0.05). To characterize direct targets of tHBxΔ35 that confer oncogenic properties, chromatin immunoprecipitation (ChIP) coupled with human promoter arrays were performed. These analyses identified 347 and 485 target genes in tHBxΔ35-expressing MIHA and LO2 cells (p<0.001), respectively, of which 42 genes were common in both cell lines. Notably, gene ontology analysis of these target genes revealed an enrichment of negative regulators of cell proliferation (p<0.005). Quantitative ChIP- and RT-PCR analyses further demonstrated that one of the common target genes, growth arrest specific 2 (GAS2) was directly repressed by tHBxΔ35 in liver cells and down-regulated in a subset (22/54) of HBV-associated HCCs compared with the non-tumor tissues. Ectopic GAS2 expression in SK-Hep1 HCC cells significantly decreased cell growth by MTS and colony formation assays (p<0.05). The GAS2-induced growth inhibition was mediated by apoptosis as shown by increase in sub-G1 population and induction of caspase-3 and PARP cleavage. Treatment with a chemotherapy drug etoposide further enhanced the apoptotic effect of GAS2. Conversely, siRNA-mediated knockdown of GAS2 and etoposide treatment synergistically decreased caspase-3 and PARP cleavage in MIHA and HepG2 cells. Furthermore, TF binding site analysis revealed highly significant over-representation of POU3F2 binding sites in tHBxΔ35-bound loci (p<1E-53) including the GAS2 promoter. In conclusion, our genome-wide binding analysis of tHBx revealed direct repression of GAS2, which may provide a selective growth advantage for precancerous cells. POU3F2 may function as a previously unidentified TF partner of tHBx in exerting its oncogenic properties. This study was supported by RFCID 08070332.
871 Reduced Jmjd3 Expression Enhances Aggressiveness of Pancreatic Cancer Through Downregulation of C/EBPα Keisuke Yamamoto, Keisuke Tateishi, Koji Miyabayashi, Shinzo Yamamoto, Yotaro Kudo, Dai Mohri, Yoshinari Asaoka, Hideaki Ijichi, Masao Omata, Kazuhiko Koike Gene transcription is influenced by chromatin structure, which is regulated partly through modification of histone tails. Recently, an increasing number of histone modifiers have been found to be deregulated in cancers, and it has been proposed that these misregulation of histone modification, through the disturbance of gene expression, may contribute to tumorigenesis and cancer progression. Trimethylation on histone H3 lysine 27 (H3K27me3) is an epigenetic mark which represses key developmental regulators in embryonic stem cells (ESCs) and tumor suppressors through the binding of polycomb group (PcG) proteins, and several H3K27me3 modulators have been reported to be deregulated in cancers. Jmjd3 is a H3K27me3 demethylase, which is proposed to suppress tumor initiation from its capacity to activate INK4a/ARF locus during oncogene-induced senescence. However, no clear relationship has been reported so far between its loss of function and tumor progression. To elucidate the role of Jmjd3 in established cancer, we first knocked down Jmjd3 in pancreatic cancer cells. We found that Jmjd3 knockdown cells were more invasive than control cells In Vitro, and that these cells showed a remarkable increase in their ability to form tumor spheres, a property associated with tumorigenicity. To confirm these phenotypic changes In Vivo, we performed xenotransplantation of these cells using nude mice. We compared the prognosis of mice injected with Jmjd3-knockdown or control cells into the spleen. Mice injected with Jmjd3 knockdown cells showed significantly poorer survival. We also performed an orthotopic implantation assay. Seven weeks after intrapancreatic implantation, only mice injected with Jmjd3 knockdown cells developed massive peritoneal dissemination with hemorrhagic ascites, while no mice injected with control cells developed peritoneal dissemination. To identify genes responsible for these phenotypic change, we performed cDNA microarray analysis. Gene set enrichment analysis revealed that the expression of C/EBPα, a putative tumor suppressor known to be downregulated in various cancers, and its target genes were significantly changed upon Jmjd3 knockdown. Futhermore, chromatin immunoprecipitation analysis revealed that H3K27me3 levels in regions close to the transcription start site of C/ EBPα gene were specifically increased in response to Jmjd3 knockdown and correlated negatively with C/EBPα expression. Moreover, we also found that enforced expression of C/EBPα rescued the increased invasiveness and tumorigenicity of Jmjd3 knockdown cells. These results indicate that reduction of Jmjd3 expression in pancreatic cancer enhances aggressiveness through downregulation of C/EBPα.
869 Loss of the Notch-ATOH1 Regulated Differentiation Factor, SPDEF, Increases Colon Tumor Number and Induces Local Invasion Taeko K. Noah, Chelsea D. Tolentino, Eli Marr, Noah F. Shroyer Background: We recently identified that SPDEF (SAM Pointed domain ETS Transcription Factor, also called PDEF) expression is significantly reduced in the majority of human colon cancers. This reduction of SPDEF expression was tightly correlated with loss of the Notch regulated pro-secretory factor, Atoh1, which we reported to function as a tumor suppressor. Other In Vitro studies suggest that SPDEF is a tumor suppressor in colon, prostate and breast cancers involving in regulation of migration and invasion. Here, we tested the hypothesis that SPDEF functions as a tumor suppressor in mouse colon. Method: Intestinal polyps were induced in SPDEF knockout (KO) mice with three methods; (1) azoxymethane and dextran sodium sulfate (AOM+DSS),(2) dimethylhydrazine and dextran sodium sulfate (DMH+DSS) and (3) ApcMin/+ mutation. Polyp number was quantified in AOM+DSS and ApcMin/+ models. DMH+DSS model was used to assess tumor invasion. Tumor cell proliferation and invasiveness was assessed immunohistologically. Results: Polyp number was significantly increased in SPDEF KO mice compared to wild type (WT) littermate controls with both models (AOM+DSS and ApcMin/+). Tumor cell proliferation and apoptosis was not different between SPDEF KO and WT. Interestingly, we detected local invasion in more than 50% of SPDEF KO tumors from mice treated with DMH+DSS. No local invasion was observed in wild type littermate controls treated with DMH+DSS. Conclusions: SPDEF is a tumor suppressor in intestinal epithelia that inhibits both tumor formation and invasion. Our results suggest that SPDEF is a functional target for therapeutic intervention in colon cancer.
872 Epigenetic Characterization of RAS Association Domain-Containing Protein 10 as a Functional Tumor Suppressor in Gastric Cancer Xiaoxing Li, Weili Liu, Kin-Fai Cheung, Eagle SH Chu, Qian Tao, Joseph J. Sung, Jun Yu Background & Aims: The ras association domain family (RASSF) encodes for tumor suppressors with several members being frequently epigenetically inactivated in cancer. RASSF10 locates chromosome 11p15.2 and encodes a 507 amino acids protein. However, the role of RASSF10 in cancer has remained largely unknown. In this study, we analyzed epigenetic inactivation and biological functions of RASSF10 in gastric cancer. Methods: Expression level of RASSF10 was detected by reverse transcription-PCR. Methylation status of RASSF10 in 16 gastric cancer cell lines and 117 gastric cancer biopsies was evaluated by methylation specific-PCR (MS-PCR), combined bisulfite restriction analysis (COBRA) and bisulfite genomic sequencing (BGS). The biological functions of RASSF10 re-expression in silenced gastric cancer cell lines were determined by cell proliferation, apoptosis, migration and invasion assays. Expression array was performed to reveal the molecular basis of RASSF10 function in gastric cancer. Results: RASSF10 was silenced or down-regulated in 56% (9 of 16) of gastric cancer cell lines. In contract, RASSF10 is broadly expressed in digestive organs including esophagus, stomach and colon. The silence or downregulation of RASSF10 in gastric cancer cell lines is closely related to the CpG sites hypermethylation status in the promoter of RASSF10 gene as determined by COBRA and BGS analyses. Expression of
870 Protein Tyrosine Kinase 6 Expression is Regulated by p53-Dependent and Independent Mechanisms in Response to DNA-Damage Jessica Gierut, Ansu O. Perekatt, Angela L. Tyner BACKGROUND & AIMS: Protein Tyrosine Kinase 6 (PTK6) is an intracellular tyrosine kinase that promotes epithelial cell differentiation and cell cycle exit in the normal intestine. However, following DNA damage, PTK6 is induced in proliferating crypt epithelial cells in the small intestine and colon where it promotes apoptosis by inhibiting prosurvival signaling pathways. The tumor suppressor protein p53, which is frequently mutated in colon cancer, is a transcription factor that is induced after DNA damage. The aim of our study was to determine if p53 regulates induction of PTK6 expression following DNA damage, and if
AGA Abstracts
S-144
904 TELAPREVIR-BASED REGIMENS SUBSTANTIALLY IMPROVED SVR RATES ACROSS ALL IL28B GENOTYPES IN THE ADVANCE TRIAL Ira M. Jacobson, Ian M. Catlett, Patrick Marcellin, Natalie H. Bzowej, Andrew J. Muir, Nathalie Adda, Shelia Seepersaud, Ravi Ramachandran, Katherine Sussky, Leif Bengtsson, Shelley George, Robert S. Kauffman, Martyn C. Botfield
873 Paired Box Gene 5 is a Novel Tumor Suppressor Involved in the Pathogenesis of Hepatocellular Carcinoma Through Interaction With p53 Signaling Pathway Weili Liu, Xiaoxing Li, Eagle SH Chu, Minnie Y. Go, Joseph J. Sung, Jun Yu
Background & Aim: Single nucleotide polymorphisms (SNPs) near the IL28B gene have been strongly associated with the likelihood of SVR in genotype 1 HCV patients treated with peginterferon/ribavirin (PR). During the evaluation of an exploratory diagnostic test that characterizes genetic polymorphisms near the IL28B gene, the impact of rs12979860 on SVR in telaprevir (T)-based regimens was evaluated. Methods: IL28B genotype was determined in de-identified left-over specimens available from treatment-naïve genotype 1 HCV patients from ADVANCE U.S. sites. Because of the limited number of patients of nonCaucasian race and the requirements of the de-identification procedure, only samples from Caucasian patients were tested. Results: The diagnostic assay developed provided consistent, unambiguous genotype calls and was considered suitable for research. 454/1088 (42%) patients had IL28B test results available. 150/454 (33%) were CC, 224/454 (49%) CT, and 80/454 (18%) TT. SVR rates for each subgroup by arm are shown in the Table. 72%, 54% and 48% of CC, CT and TT telaprevir patients, respectively had undetectable HCV RNA at weeks 4 and 12 (eRVR) compared with 16%, 3% and 0% of PR patients. Among eRVR telaprevir patients, 91% achieved SVR (97% of CC, 88% of CT/TT) with 24 weeks of therapy whereas 45% of non-eRVR telaprevir patients had SVR (67% of CC, 38% CT/TT) with 48 weeks of therapy. Conclusions: Telaprevir-based therapy improved eRVR and SVR rates across all IL28B genotypes. Specifically, telaprevir-based therapy more than doubled the rates of SVR in CT/TT patients, and substantially increased SVR rates in those with CC genotype, as compared with PR therapy alone. Non-attainment of eRVR was associated with lower SVR rates across all IL28B genotypes, with the largest decrement in CT/TT patients. SVR rates
Background: The paired box 5 (PAX5) is a member of PAX transcription factors family associated with the regulation of embryonic development, however the role of PAX5 in carcinogenesis is largely unclear. We identified PAX5 is involved in human cancer by methylation-sensitive representational difference analysis. We analyzed the epigenetic inactivation, biological functions and related molecular mechanisms of PAX5 in hepatocellular carcinoma (HCC). Methods: Promoter methylation of PAX5 was evaluated by methylationspecific PCR and bisulfite genomic sequencing (BGS). The functions of ectopic PAX5 expressions were determined by viability assays, colony formation and cell cycle analyses, along with In Vivo tumorigenicity assay. PAX5 target signal pathway was identified by promoter luciferase assay, chromosome immunoprecipitation (ChIP) and pathway PCR array analyses. Results: PAX5 is expressed in normal human liver tissue, but silenced or down-regulated in 83% (10/12) of HCC cell lines. Real-time PCR analysis showed that PAX5 expression is significantly down-regulated in primary HCCs as compared with their corresponding adjacent normal tissues (P=0.0008). The promoter methylation status contributes to the inactivity of PAX5 in HCC cell lines, as evidenced by demethylating treatment and BGS. Restoring PAX5 expression in Hep3B or HepG2 HCC cell line suppressed cell viability (P<0.0001), colony formation (down to 44-54% of vector control, P<0.01), induced apoptosis (24.75% ± 2.09% vs., 33.11% ± 2.06%, P<0.05) In Vitro, and inhibited tumor growth in nude mice (P<0.0001). Pathway luciferase reporter assays indicated that PAX5 activated p53 and p21 signalings. ChIP assay demonstrated that PAX5 directly bound to the p53 promoter. The anti-tumorigenic function of PAX5 were at least by up-regulating p53 and its downstream targets including tumor necrosis factor, Fas ligand, leucine-rich repeats and death domain containing, poly(rC) binding protein4, p21 and growth arrest and DNA-damage-inducible, alpha. Conclusions: PAX5 is frequently inactivated by promoter methylation in HCCs. PAX5 appears to be a functional tumor suppressor involved in liver carcinogenesis through directly regulating p53 signaling pathway. Acknowledgment The project was supported by Research Grants Council Competitive Earmarked Research Grant CUHK (GRF 473008, GRF 478108, CUHK/CRF/ 09 and scheme B MK/KT/fis0910/0543/10yc).
*T12PR = T+PR 12 weeks, then PR 12 or 36 weeks depending on eRVR status **T8PR = T+PR 8 weeks, then PR 16 or 40 weeks depending on eRVR status §In patients tested for IL28B, SVR was defined as undetectable HCV RNA at EOT and at 24 weeks after last actual dose of study drug †In the ADVANCE study, SVR was defined as undetectable HCV RNA at EOT and at 24 weeks after last planned dose of study drug
903 High Sustained Virologic Response (SVR) Among Genotype 1 Previous NonResponders and Relapsers to Peginterferon/Ribavirin When Re-Treated with Boceprevir (BOC) Plus Peginterferon Alfa-2A/Ribavirin Steven L. Flamm, Eric J. Lawitz, Ira M. Jacobson, Raymond A. Rubin, Marc Bourliere, C. Hezode, J. Vierling, Claus Niederau, Morris Sherman, Venkata S. Goteti, Regis A. Vilchez, Clifford A. Brass, Janice K. Albrecht, Fred Poordad
905 Obtaining SVR To Antiviral Therapy Highly Protects Patients With HCV Recurrence On The Graft After Liver Transplantation From Liver Related Death: Insights from The AISF-RECOLT-C GROUP. Maria Rendina, Nicola M. Castellaneta, Stefano Fagiuoli, Francesca R. Ponziani, Chiara Vigano', Rosa Maria Iemmolo, Maria F. Donato, Pierluigi Toniutto, Luisa Pasulo, Maria Cristina Morelli, Patrizia Burra, Lucia Miglioresi, Valerio Giannelli, Daniele Di Paolo, Alfredo Di Leo
Background: The RESPOND-2 trial demonstrated significantly increased SVR for prior nonresponders and relapsers when BOC was added to peginterferon alfa-2b/ribavirin (66% vs. 21% control). We assessed SVR with BOC combined with peginterferon alfa-2a (PEG2a) and ribavirin (R) in patients who met identical entry criteria. Methods: This double-blind, placebo-controlled trial randomized 201 genotype-1 relapsers and non-responders to two arms (1:2 ratio, Table). Arm 1 (control) received a 4-week lead-in of PEG2a/R followed by placebo + PEG2a/R for 44 weeks. Arm 2 received a 4-week lead-in of PEG2a/R followed by BOC + PEG2a/R for 44 weeks. Therapy was discontinued if HCV-RNA was detectable (undetectable HCV RNA <9.3 IU/mL [Roche TaqMan, LLD]) at week 12. Primary endpoint: SVR 24-weeks post-therapy. Results: Baseline demographics: 70% male; 10% black; 16% cirrhotic. The addition of BOC after a 4-week lead-in with PEG2a/R significantly increased SVR: 21% in Arm 1 vs. 64% in Arm 2 (p<0.0001). SVR for patients with poor interferon responsiveness (<1-log10 decrease in HCV-RNA after 4-week lead-in) was 0% in Arm 1 and 39% in Arm 2. For patients responsive to interferon (≥1-log10 decrease in HCV-RNA after 4-week lead-in), SVR was 25% in Arm 1 and 71% in Arm 2. Discontinuation due to adverse events (AEs) occurred in 4% and 17% of patients in Arms 1 and 2. Rates of serious AEs were 10% in Arm 1 and 13% in Arm 2. The frequencies of anemia (<10.0 g/dL) in Arms 1 and 2 were 27% vs. 49%; neutropenia (WHO grade 3-4 [<750/mm3]) 21% vs. 43%; erythropoietin use 30% vs. 47%. There were no serious AEs due to anemia and one discontinuation due to anemia (Arm 2). Conclusions: Lead-in with PEG2a and ribavirin followed by addition of boceprevir resulted in high SVR rates similar to that observed using an identical treatment regimen with peginterferon alfa-2b. Therapy was generally welltolerated. These are the first large trials to demonstrate a direct acting antiviral agent may be combined with either PEG2a or PEG2b to significantly increase SVR in patients who failed prior therapy.
Background: Results of antiviral therapy for HCV recurrence on the graft are controversial. Sustained virological response (SVR) to antiviral is strongly associated with reduced liverrelated mortality in chronic hepatitis and cirrhosis. In liver transplant (LT) setting, however, a huge number of factors and co-morbidities could frustrate the benefit provided by the elimination of the virus. Aim of the Study: To determine the impact of achieving SVR on liver related and non-liver related mortality in HCV recurrent pts, analysis was made of a large retrospective multicentre database (from 12 LT Centres in Italy. Patients and methods: Data on 464 pts (transplant between 1989 and 2008) were retrospectively analyzed. All pts, after virological and histological diagnosis of HCV recurrence, underwent antiviral therapy with standard or peginterferon (24% and 76%) plus ribavirin . Mean age at LT 53.7±7.9 yrs; male/female: 348/116; genotype 1-4: 75% pts; diabetes: 50%. Mean interval from LT to therapy: 24±28 months; median duration of therapy: 44.8±35 weeks. Results: Overall, the SVR rate was 35% (164/464). SVR pts had: younger donor age (p< 0.009), longer treatment duration and higher cumulative IFN dose (p<0.002), lower drop-out rate and less diabetes (p<0.001). No differences in immunosuppression. During post-treatment F-up of 4.4 years 120 patients died or were re-transplanted. Mortality rate in SVR and non-SVR pts was 10% and 34% respectively (p< 0.001). The incidence rate of liver-related mortality/100 persons/year of follow up was significantly different between SVR and no-SVR (0.1 vs 6.3, p< 0.001); no differences in the non-liver related mortality (1.8 vs 1.4; P 0.4). At univariate Cox regression, risk factors for death/re-transplantation were older donor age (P<0.001; HR 1.03), diabetes (P< 0.001; HR 2.3), genotype 1-4 (P 0.03; HR 1.7) and lack of SVR (P< 0.001; HR 3.9). At multivariate analysis, failure in obtaining SVR remained significantly associated with mortality (HR 3.4 p=0.001) Conclusion: Despite significant drawbacks in conducting antiviral therapy in HCV recurrent liver transplant patients, strategies of HCV
S-145
AGA Abstracts
AGA Abstracts
RASSF10 in silenced gastric cancer cell lines could be restored after treating with demethylation agent, 5-aza-deoxycytidine. We further tested the biological function of RASSF10 in human gastric cancer cells. Stable transfection of RASSF10 in AGS and MKN45 cancer cells reduced colony formation from 100% to 51.6% in AGS (P<0.01) and to 56.0% in MKN45 (P<0.01). Ectopic expression of RASSF10 in AGS and MKN45 cells significantly suppressed cell viability (P<0.0001 for both cell lines), induced cell apoptosis (P<0.05 for both cell lines), and repressed cell migration and invasion (P<0.0001 for both cell lines). We further found that RASSF10 up-regulated the expression of pro-apoptotic gene TNF and metastasis suppressor MTSS1, and down-regulated the expression of multiple oncogenes including AKT1, ERBB2, FOS and Jun. RASSF10 hypermethylation was detected in 54% (53/99) of primary gastric cancers, but only in 6% (1/18) of non-cancer tissues (P<0.0001). Conclusions: Our data show that RASSF10 is a functional tumor suppressor frequently methylated in gastric cancers. Acknowledgment The project was supported by Research Grant ERG CUHK (GRF 473008).
disease. TLR2-/-MyD88+/+MDR1A-/- macrophages hyperresponded to LPS stimulation with increased production of IL-1β protein which was not evident in TLR2+/+MyD88+/+MDR1A-/macrophages. Conclusions: Exacerbation of UC-like pancolitis in TLR2/MDR1A double deficiency requires IL-1R/MyD88 signalling. Aberrant LPS-induced IL-1β signalling via IL1R/MyD88 may represent a critical inducer of severe pathology and a novel therapeutic target in a subgroup of UC patients.
PTK6 modulates colon cancer cell sensitivity to chemotherapeutic agents. METHODS: Isogenic HCT116 p53+/+ and p53-/- human colon cancer cell lines containing empty vector or expressing a scrambled shRNA or two different shRNAs that target PTK6 were treated with doxorubicin and 5-fluorouracil, drugs used in cancer chemotherapy. The cell lines were also subjected to 20 Gy of gamma-irradiation and harvested at 0, 3, 6, 24 and 48 hours. Protein lysates were prepared and analyzed by immunoblotting. Cells were incubated with PE Annexin V and 7-Amino-Actinomycin and apoptosis was analyzed by flow cytometry. RNA was prepared and expression of PTK6 and the p53 target gene p21 was examined by qRT-PCR. RESULTS: Stable knockdown of PTK6 in HCT116 p53+/+ and p53-/- cells using two different shRNAs led to increased apoptosis in both HCT116 p53+/+ and HCT116 p53-/- cells with a more dramatic increase in HCT116 p53-/- cells following treatment with doxorubicin, 5-fluorouracil, and radiation. Highest levels of cleaved caspase-3 and apoptosis were detected in PTK6 negative p53-/- cells using immunoblotting and flow cytometry. Rapid accumulation of p53 and activation of the p53 target gene encoding p21 was detected in wild type HCT116 cells following all treatments. Induction of PTK6 was observed by 24 hours after irradiation, with highest levels at 48 hours post irradiation. While PTK6 was induced in both HCT116 p53+/+ and p53-/- cell lines following irradiation, higher levels of PTK6 protein and mRNA were detected in p53+/+ HCT116 cells suggesting a p53dependent component in PTK6 induction. However unlike the p21 gene that is induced by 3 hours post irradiation, the PTK6 gene does not appear to be a direct target of p53. Robust induction of PTK6 was not evident after treatment of HCT116 cells with either doxorubicin or 5-fluorouracil. CONCLUSIONS: PTK6 expression is positively regulated by p53-dependent and independent mechanisms after DNA damage. Knockdown of PTK6 expression promotes apoptosis of HCT116 cells and strongly enhances the response of p53-/colon tumor cells to irradiation, doxorubicin and 5-fluorouracil. Kinase inhibitors targeting PTK6 may enhance sensitivity of colon cancer cells to chemotherapeutic agents.
868 Genome-Wide Binding Analysis of Carboxyl-Terminal Truncated HBx Reveals Direct Repression of a Negative Growth Regulator, GAS2, in Hepatocellular Carcinoma Ranxu Zhu, Alfred S. Cheng, Suki S. Lau, Yangchao Chen, Tin L Lee, Vincent W. Wong, Joseph J. Sung, Henry Lik-Yuen Chan Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) and the X protein of HBV (HBx) is highly implicated in hepatocarcinogenesis. Recently HBx has been shown to act as coregulator of cancer-related genes by interacting with DNAbound transcription factor (TF). The carboxyl-terminal truncated forms of HBx (tHBx), rather than the full-length counterpart, have been shown to frequently express in HCC tissues and possess oncogenic abilities In Vitro and In Vivo. tHBxΔ35 (deletion of 35 carboxylamino acids) is one of the truncated oncoproteins naturally generated by cytidine deaminases in HCC tissues. However, how tHBxΔ35 exerts oncogenic effects is poorly understood. Here we used genome-wide location analysis and functional studies to elucidate the role of direct tHBxΔ35 target genes in the development of HCC. tHBxΔ35 was cloned from the serum of an HCC patient infected with HBV genotype C. Ectopic expression of tHBxΔ35 in 2 immortal human liver cell lines (MIHA and LO2) significantly increased cell proliferation compared with vector-control cells as shown by MTS and colony formation assays (p<0.05). To characterize direct targets of tHBxΔ35 that confer oncogenic properties, chromatin immunoprecipitation (ChIP) coupled with human promoter arrays were performed. These analyses identified 347 and 485 target genes in tHBxΔ35-expressing MIHA and LO2 cells (p<0.001), respectively, of which 42 genes were common in both cell lines. Notably, gene ontology analysis of these target genes revealed an enrichment of negative regulators of cell proliferation (p<0.005). Quantitative ChIP- and RT-PCR analyses further demonstrated that one of the common target genes, growth arrest specific 2 (GAS2) was directly repressed by tHBxΔ35 in liver cells and down-regulated in a subset (22/54) of HBV-associated HCCs compared with the non-tumor tissues. Ectopic GAS2 expression in SK-Hep1 HCC cells significantly decreased cell growth by MTS and colony formation assays (p<0.05). The GAS2-induced growth inhibition was mediated by apoptosis as shown by increase in sub-G1 population and induction of caspase-3 and PARP cleavage. Treatment with a chemotherapy drug etoposide further enhanced the apoptotic effect of GAS2. Conversely, siRNA-mediated knockdown of GAS2 and etoposide treatment synergistically decreased caspase-3 and PARP cleavage in MIHA and HepG2 cells. Furthermore, TF binding site analysis revealed highly significant over-representation of POU3F2 binding sites in tHBxΔ35-bound loci (p<1E-53) including the GAS2 promoter. In conclusion, our genome-wide binding analysis of tHBx revealed direct repression of GAS2, which may provide a selective growth advantage for precancerous cells. POU3F2 may function as a previously unidentified TF partner of tHBx in exerting its oncogenic properties. This study was supported by RFCID 08070332.
871 Reduced Jmjd3 Expression Enhances Aggressiveness of Pancreatic Cancer Through Downregulation of C/EBPα Keisuke Yamamoto, Keisuke Tateishi, Koji Miyabayashi, Shinzo Yamamoto, Yotaro Kudo, Dai Mohri, Yoshinari Asaoka, Hideaki Ijichi, Masao Omata, Kazuhiko Koike Gene transcription is influenced by chromatin structure, which is regulated partly through modification of histone tails. Recently, an increasing number of histone modifiers have been found to be deregulated in cancers, and it has been proposed that these misregulation of histone modification, through the disturbance of gene expression, may contribute to tumorigenesis and cancer progression. Trimethylation on histone H3 lysine 27 (H3K27me3) is an epigenetic mark which represses key developmental regulators in embryonic stem cells (ESCs) and tumor suppressors through the binding of polycomb group (PcG) proteins, and several H3K27me3 modulators have been reported to be deregulated in cancers. Jmjd3 is a H3K27me3 demethylase, which is proposed to suppress tumor initiation from its capacity to activate INK4a/ARF locus during oncogene-induced senescence. However, no clear relationship has been reported so far between its loss of function and tumor progression. To elucidate the role of Jmjd3 in established cancer, we first knocked down Jmjd3 in pancreatic cancer cells. We found that Jmjd3 knockdown cells were more invasive than control cells In Vitro, and that these cells showed a remarkable increase in their ability to form tumor spheres, a property associated with tumorigenicity. To confirm these phenotypic changes In Vivo, we performed xenotransplantation of these cells using nude mice. We compared the prognosis of mice injected with Jmjd3-knockdown or control cells into the spleen. Mice injected with Jmjd3 knockdown cells showed significantly poorer survival. We also performed an orthotopic implantation assay. Seven weeks after intrapancreatic implantation, only mice injected with Jmjd3 knockdown cells developed massive peritoneal dissemination with hemorrhagic ascites, while no mice injected with control cells developed peritoneal dissemination. To identify genes responsible for these phenotypic change, we performed cDNA microarray analysis. Gene set enrichment analysis revealed that the expression of C/EBPα, a putative tumor suppressor known to be downregulated in various cancers, and its target genes were significantly changed upon Jmjd3 knockdown. Futhermore, chromatin immunoprecipitation analysis revealed that H3K27me3 levels in regions close to the transcription start site of C/ EBPα gene were specifically increased in response to Jmjd3 knockdown and correlated negatively with C/EBPα expression. Moreover, we also found that enforced expression of C/EBPα rescued the increased invasiveness and tumorigenicity of Jmjd3 knockdown cells. These results indicate that reduction of Jmjd3 expression in pancreatic cancer enhances aggressiveness through downregulation of C/EBPα.
869 Loss of the Notch-ATOH1 Regulated Differentiation Factor, SPDEF, Increases Colon Tumor Number and Induces Local Invasion Taeko K. Noah, Chelsea D. Tolentino, Eli Marr, Noah F. Shroyer Background: We recently identified that SPDEF (SAM Pointed domain ETS Transcription Factor, also called PDEF) expression is significantly reduced in the majority of human colon cancers. This reduction of SPDEF expression was tightly correlated with loss of the Notch regulated pro-secretory factor, Atoh1, which we reported to function as a tumor suppressor. Other In Vitro studies suggest that SPDEF is a tumor suppressor in colon, prostate and breast cancers involving in regulation of migration and invasion. Here, we tested the hypothesis that SPDEF functions as a tumor suppressor in mouse colon. Method: Intestinal polyps were induced in SPDEF knockout (KO) mice with three methods; (1) azoxymethane and dextran sodium sulfate (AOM+DSS),(2) dimethylhydrazine and dextran sodium sulfate (DMH+DSS) and (3) ApcMin/+ mutation. Polyp number was quantified in AOM+DSS and ApcMin/+ models. DMH+DSS model was used to assess tumor invasion. Tumor cell proliferation and invasiveness was assessed immunohistologically. Results: Polyp number was significantly increased in SPDEF KO mice compared to wild type (WT) littermate controls with both models (AOM+DSS and ApcMin/+). Tumor cell proliferation and apoptosis was not different between SPDEF KO and WT. Interestingly, we detected local invasion in more than 50% of SPDEF KO tumors from mice treated with DMH+DSS. No local invasion was observed in wild type littermate controls treated with DMH+DSS. Conclusions: SPDEF is a tumor suppressor in intestinal epithelia that inhibits both tumor formation and invasion. Our results suggest that SPDEF is a functional target for therapeutic intervention in colon cancer.
872 Epigenetic Characterization of RAS Association Domain-Containing Protein 10 as a Functional Tumor Suppressor in Gastric Cancer Xiaoxing Li, Weili Liu, Kin-Fai Cheung, Eagle SH Chu, Qian Tao, Joseph J. Sung, Jun Yu Background & Aims: The ras association domain family (RASSF) encodes for tumor suppressors with several members being frequently epigenetically inactivated in cancer. RASSF10 locates chromosome 11p15.2 and encodes a 507 amino acids protein. However, the role of RASSF10 in cancer has remained largely unknown. In this study, we analyzed epigenetic inactivation and biological functions of RASSF10 in gastric cancer. Methods: Expression level of RASSF10 was detected by reverse transcription-PCR. Methylation status of RASSF10 in 16 gastric cancer cell lines and 117 gastric cancer biopsies was evaluated by methylation specific-PCR (MS-PCR), combined bisulfite restriction analysis (COBRA) and bisulfite genomic sequencing (BGS). The biological functions of RASSF10 re-expression in silenced gastric cancer cell lines were determined by cell proliferation, apoptosis, migration and invasion assays. Expression array was performed to reveal the molecular basis of RASSF10 function in gastric cancer. Results: RASSF10 was silenced or down-regulated in 56% (9 of 16) of gastric cancer cell lines. In contract, RASSF10 is broadly expressed in digestive organs including esophagus, stomach and colon. The silence or downregulation of RASSF10 in gastric cancer cell lines is closely related to the CpG sites hypermethylation status in the promoter of RASSF10 gene as determined by COBRA and BGS analyses. Expression of
870 Protein Tyrosine Kinase 6 Expression is Regulated by p53-Dependent and Independent Mechanisms in Response to DNA-Damage Jessica Gierut, Ansu O. Perekatt, Angela L. Tyner BACKGROUND & AIMS: Protein Tyrosine Kinase 6 (PTK6) is an intracellular tyrosine kinase that promotes epithelial cell differentiation and cell cycle exit in the normal intestine. However, following DNA damage, PTK6 is induced in proliferating crypt epithelial cells in the small intestine and colon where it promotes apoptosis by inhibiting prosurvival signaling pathways. The tumor suppressor protein p53, which is frequently mutated in colon cancer, is a transcription factor that is induced after DNA damage. The aim of our study was to determine if p53 regulates induction of PTK6 expression following DNA damage, and if
AGA Abstracts
S-144
904 TELAPREVIR-BASED REGIMENS SUBSTANTIALLY IMPROVED SVR RATES ACROSS ALL IL28B GENOTYPES IN THE ADVANCE TRIAL Ira M. Jacobson, Ian M. Catlett, Patrick Marcellin, Natalie H. Bzowej, Andrew J. Muir, Nathalie Adda, Shelia Seepersaud, Ravi Ramachandran, Katherine Sussky, Leif Bengtsson, Shelley George, Robert S. Kauffman, Martyn C. Botfield
873 Paired Box Gene 5 is a Novel Tumor Suppressor Involved in the Pathogenesis of Hepatocellular Carcinoma Through Interaction With p53 Signaling Pathway Weili Liu, Xiaoxing Li, Eagle SH Chu, Minnie Y. Go, Joseph J. Sung, Jun Yu
Background & Aim: Single nucleotide polymorphisms (SNPs) near the IL28B gene have been strongly associated with the likelihood of SVR in genotype 1 HCV patients treated with peginterferon/ribavirin (PR). During the evaluation of an exploratory diagnostic test that characterizes genetic polymorphisms near the IL28B gene, the impact of rs12979860 on SVR in telaprevir (T)-based regimens was evaluated. Methods: IL28B genotype was determined in de-identified left-over specimens available from treatment-naïve genotype 1 HCV patients from ADVANCE U.S. sites. Because of the limited number of patients of nonCaucasian race and the requirements of the de-identification procedure, only samples from Caucasian patients were tested. Results: The diagnostic assay developed provided consistent, unambiguous genotype calls and was considered suitable for research. 454/1088 (42%) patients had IL28B test results available. 150/454 (33%) were CC, 224/454 (49%) CT, and 80/454 (18%) TT. SVR rates for each subgroup by arm are shown in the Table. 72%, 54% and 48% of CC, CT and TT telaprevir patients, respectively had undetectable HCV RNA at weeks 4 and 12 (eRVR) compared with 16%, 3% and 0% of PR patients. Among eRVR telaprevir patients, 91% achieved SVR (97% of CC, 88% of CT/TT) with 24 weeks of therapy whereas 45% of non-eRVR telaprevir patients had SVR (67% of CC, 38% CT/TT) with 48 weeks of therapy. Conclusions: Telaprevir-based therapy improved eRVR and SVR rates across all IL28B genotypes. Specifically, telaprevir-based therapy more than doubled the rates of SVR in CT/TT patients, and substantially increased SVR rates in those with CC genotype, as compared with PR therapy alone. Non-attainment of eRVR was associated with lower SVR rates across all IL28B genotypes, with the largest decrement in CT/TT patients. SVR rates
Background: The paired box 5 (PAX5) is a member of PAX transcription factors family associated with the regulation of embryonic development, however the role of PAX5 in carcinogenesis is largely unclear. We identified PAX5 is involved in human cancer by methylation-sensitive representational difference analysis. We analyzed the epigenetic inactivation, biological functions and related molecular mechanisms of PAX5 in hepatocellular carcinoma (HCC). Methods: Promoter methylation of PAX5 was evaluated by methylationspecific PCR and bisulfite genomic sequencing (BGS). The functions of ectopic PAX5 expressions were determined by viability assays, colony formation and cell cycle analyses, along with In Vivo tumorigenicity assay. PAX5 target signal pathway was identified by promoter luciferase assay, chromosome immunoprecipitation (ChIP) and pathway PCR array analyses. Results: PAX5 is expressed in normal human liver tissue, but silenced or down-regulated in 83% (10/12) of HCC cell lines. Real-time PCR analysis showed that PAX5 expression is significantly down-regulated in primary HCCs as compared with their corresponding adjacent normal tissues (P=0.0008). The promoter methylation status contributes to the inactivity of PAX5 in HCC cell lines, as evidenced by demethylating treatment and BGS. Restoring PAX5 expression in Hep3B or HepG2 HCC cell line suppressed cell viability (P<0.0001), colony formation (down to 44-54% of vector control, P<0.01), induced apoptosis (24.75% ± 2.09% vs., 33.11% ± 2.06%, P<0.05) In Vitro, and inhibited tumor growth in nude mice (P<0.0001). Pathway luciferase reporter assays indicated that PAX5 activated p53 and p21 signalings. ChIP assay demonstrated that PAX5 directly bound to the p53 promoter. The anti-tumorigenic function of PAX5 were at least by up-regulating p53 and its downstream targets including tumor necrosis factor, Fas ligand, leucine-rich repeats and death domain containing, poly(rC) binding protein4, p21 and growth arrest and DNA-damage-inducible, alpha. Conclusions: PAX5 is frequently inactivated by promoter methylation in HCCs. PAX5 appears to be a functional tumor suppressor involved in liver carcinogenesis through directly regulating p53 signaling pathway. Acknowledgment The project was supported by Research Grants Council Competitive Earmarked Research Grant CUHK (GRF 473008, GRF 478108, CUHK/CRF/ 09 and scheme B MK/KT/fis0910/0543/10yc).
*T12PR = T+PR 12 weeks, then PR 12 or 36 weeks depending on eRVR status **T8PR = T+PR 8 weeks, then PR 16 or 40 weeks depending on eRVR status §In patients tested for IL28B, SVR was defined as undetectable HCV RNA at EOT and at 24 weeks after last actual dose of study drug †In the ADVANCE study, SVR was defined as undetectable HCV RNA at EOT and at 24 weeks after last planned dose of study drug
903 High Sustained Virologic Response (SVR) Among Genotype 1 Previous NonResponders and Relapsers to Peginterferon/Ribavirin When Re-Treated with Boceprevir (BOC) Plus Peginterferon Alfa-2A/Ribavirin Steven L. Flamm, Eric J. Lawitz, Ira M. Jacobson, Raymond A. Rubin, Marc Bourliere, C. Hezode, J. Vierling, Claus Niederau, Morris Sherman, Venkata S. Goteti, Regis A. Vilchez, Clifford A. Brass, Janice K. Albrecht, Fred Poordad
905 Obtaining SVR To Antiviral Therapy Highly Protects Patients With HCV Recurrence On The Graft After Liver Transplantation From Liver Related Death: Insights from The AISF-RECOLT-C GROUP. Maria Rendina, Nicola M. Castellaneta, Stefano Fagiuoli, Francesca R. Ponziani, Chiara Vigano', Rosa Maria Iemmolo, Maria F. Donato, Pierluigi Toniutto, Luisa Pasulo, Maria Cristina Morelli, Patrizia Burra, Lucia Miglioresi, Valerio Giannelli, Daniele Di Paolo, Alfredo Di Leo
Background: The RESPOND-2 trial demonstrated significantly increased SVR for prior nonresponders and relapsers when BOC was added to peginterferon alfa-2b/ribavirin (66% vs. 21% control). We assessed SVR with BOC combined with peginterferon alfa-2a (PEG2a) and ribavirin (R) in patients who met identical entry criteria. Methods: This double-blind, placebo-controlled trial randomized 201 genotype-1 relapsers and non-responders to two arms (1:2 ratio, Table). Arm 1 (control) received a 4-week lead-in of PEG2a/R followed by placebo + PEG2a/R for 44 weeks. Arm 2 received a 4-week lead-in of PEG2a/R followed by BOC + PEG2a/R for 44 weeks. Therapy was discontinued if HCV-RNA was detectable (undetectable HCV RNA <9.3 IU/mL [Roche TaqMan, LLD]) at week 12. Primary endpoint: SVR 24-weeks post-therapy. Results: Baseline demographics: 70% male; 10% black; 16% cirrhotic. The addition of BOC after a 4-week lead-in with PEG2a/R significantly increased SVR: 21% in Arm 1 vs. 64% in Arm 2 (p<0.0001). SVR for patients with poor interferon responsiveness (<1-log10 decrease in HCV-RNA after 4-week lead-in) was 0% in Arm 1 and 39% in Arm 2. For patients responsive to interferon (≥1-log10 decrease in HCV-RNA after 4-week lead-in), SVR was 25% in Arm 1 and 71% in Arm 2. Discontinuation due to adverse events (AEs) occurred in 4% and 17% of patients in Arms 1 and 2. Rates of serious AEs were 10% in Arm 1 and 13% in Arm 2. The frequencies of anemia (<10.0 g/dL) in Arms 1 and 2 were 27% vs. 49%; neutropenia (WHO grade 3-4 [<750/mm3]) 21% vs. 43%; erythropoietin use 30% vs. 47%. There were no serious AEs due to anemia and one discontinuation due to anemia (Arm 2). Conclusions: Lead-in with PEG2a and ribavirin followed by addition of boceprevir resulted in high SVR rates similar to that observed using an identical treatment regimen with peginterferon alfa-2b. Therapy was generally welltolerated. These are the first large trials to demonstrate a direct acting antiviral agent may be combined with either PEG2a or PEG2b to significantly increase SVR in patients who failed prior therapy.
Background: Results of antiviral therapy for HCV recurrence on the graft are controversial. Sustained virological response (SVR) to antiviral is strongly associated with reduced liverrelated mortality in chronic hepatitis and cirrhosis. In liver transplant (LT) setting, however, a huge number of factors and co-morbidities could frustrate the benefit provided by the elimination of the virus. Aim of the Study: To determine the impact of achieving SVR on liver related and non-liver related mortality in HCV recurrent pts, analysis was made of a large retrospective multicentre database (from 12 LT Centres in Italy. Patients and methods: Data on 464 pts (transplant between 1989 and 2008) were retrospectively analyzed. All pts, after virological and histological diagnosis of HCV recurrence, underwent antiviral therapy with standard or peginterferon (24% and 76%) plus ribavirin . Mean age at LT 53.7±7.9 yrs; male/female: 348/116; genotype 1-4: 75% pts; diabetes: 50%. Mean interval from LT to therapy: 24±28 months; median duration of therapy: 44.8±35 weeks. Results: Overall, the SVR rate was 35% (164/464). SVR pts had: younger donor age (p< 0.009), longer treatment duration and higher cumulative IFN dose (p<0.002), lower drop-out rate and less diabetes (p<0.001). No differences in immunosuppression. During post-treatment F-up of 4.4 years 120 patients died or were re-transplanted. Mortality rate in SVR and non-SVR pts was 10% and 34% respectively (p< 0.001). The incidence rate of liver-related mortality/100 persons/year of follow up was significantly different between SVR and no-SVR (0.1 vs 6.3, p< 0.001); no differences in the non-liver related mortality (1.8 vs 1.4; P 0.4). At univariate Cox regression, risk factors for death/re-transplantation were older donor age (P<0.001; HR 1.03), diabetes (P< 0.001; HR 2.3), genotype 1-4 (P 0.03; HR 1.7) and lack of SVR (P< 0.001; HR 3.9). At multivariate analysis, failure in obtaining SVR remained significantly associated with mortality (HR 3.4 p=0.001) Conclusion: Despite significant drawbacks in conducting antiviral therapy in HCV recurrent liver transplant patients, strategies of HCV
S-145
AGA Abstracts
AGA Abstracts
RASSF10 in silenced gastric cancer cell lines could be restored after treating with demethylation agent, 5-aza-deoxycytidine. We further tested the biological function of RASSF10 in human gastric cancer cells. Stable transfection of RASSF10 in AGS and MKN45 cancer cells reduced colony formation from 100% to 51.6% in AGS (P<0.01) and to 56.0% in MKN45 (P<0.01). Ectopic expression of RASSF10 in AGS and MKN45 cells significantly suppressed cell viability (P<0.0001 for both cell lines), induced cell apoptosis (P<0.05 for both cell lines), and repressed cell migration and invasion (P<0.0001 for both cell lines). We further found that RASSF10 up-regulated the expression of pro-apoptotic gene TNF and metastasis suppressor MTSS1, and down-regulated the expression of multiple oncogenes including AKT1, ERBB2, FOS and Jun. RASSF10 hypermethylation was detected in 54% (53/99) of primary gastric cancers, but only in 6% (1/18) of non-cancer tissues (P<0.0001). Conclusions: Our data show that RASSF10 is a functional tumor suppressor frequently methylated in gastric cancers. Acknowledgment The project was supported by Research Grant ERG CUHK (GRF 473008).