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Deregulated Hedgehog Signaling in the Pathogenesis of Barrett's Disease and Gastric Adenocarcinoma
node metastasis (P=0.015), higher TNM stage (P<0.0005) and distant metastasis (P=0.05) respectively. Kaplan-Meier survival curves showed that the overall survival of patients with DACT1 methylation was significantly shorter than that of patients without methylation (P=0.007). Conclusion: DACT1 acts as a functional tumor suppressor involved in gastric carcinogenesis. Acknowledgment The project was supported by Research Grant ERG CUHK (GRF 473008). 951 TFF1 Modulates NfκB Signaling and the Development of Gastric Neoplasms Mohammed Soutto, Abbes Belkhiri, M. Blanca Piazuelo, Kay Washington, Pelayo Correa, Richard M. Peek, Wael El-Rifai Background: TFF1 is a secreted protein predominantly located in the cytoplasm and surface membrane of normal gastric epithelium. Our previous report indicated a sequence of morphological changes in TFF1-knockout mice which develop progressive age-dependent carcinogenic histological events in the pyloric antrum of gastric mucosa from gastritis -> hyperplasia -> LGD -> HGD to adenocarcinoma. Methods and Results: In this study, we hypothesized that the inflammatory phenotype and molecular signature obtained in the TFF1-deficient gastric tissues are induced through activation of NFκB. NFκB plays a central role in inflammatory responses and can regulate genes associated with inflammation-dependent tumor progression processes. Using NFκB reporter assay, we found that exogenous TFF1 and stable reconstitution of TFF1 in AGS cells lead to a two-fold reduction in the NFκB reporter activity. Using qRT-PCR, we detected upregulation of several NFκB target genes (IL1b, CXCL1, CXCL2, and CXCL5) in the gastric lesions of the TFF1 KO mouse as compared to wildtype. The re-constitution of TFF1 in AGS cells led to a reduction in expression of these genes. Using Immunofluorescence assay, In Vivo, ex vivo and In Vitro studies showed that loss of TFF1 expression induced NFκB nuclear translocation whereas re-constitution of TFF1 abrogates this effect. Ex-vivo studies using epithelial cells from the pyloro-antral region of the TFF1 KO and WT mice confirmed nuclear and cytoplasmic localization of NFκBp65, respectively. In a rescue experiment, treatment of TFF1 KO cells with conditioned media from AGS-TFF1 or AGS-pcDNA cells led to an almost complete abrogation of nuclear localization of NFκB in cells treated with TFF1 conditioned media. These results confirm that loss of TFF1 activates NFκB which may represent one mechanism by which inflammation initiates gastric cancer. We examined the upstream IKKα/β complex and tested whether TFF1 may interfere with the recruitment and binding of TRAF2 to TNFR1. Using coimmunoprecipitation, our results indicated that this recruitment was abolished in AGSTFF1 cells. This finding suggests that TFF1 interference with TNFR1/TRAF2 complex is a potential mechanism of inhibiting NFκB activation. To confirm the relationship between TFF1 and NFκB in human disease, we performed immunohistochemistry analysis for NFκB and TFF1 on 100 gastric cancers including premalignant lesions. Parallel adjacent sections from the same sample demonstrated a consistent inverse relationship of the expression of these two proteins. We detected a significant progressive decrease in TFF1 expression and an increase in NFκB activation along the multi-step carcinogenesis cascade (p<.0001). Conclusion: Our findings demonstrate that loss of TFF1 plays an important role in shaping the NFκB mediated inflammatory response in the multi-step gastric tumorigenesis cascade.
949 Epigenetic Inactivation of BCL6B, a Functional Tumor Suppressor for Gastric Cancer, is Associated With Poor Survival of Gastric Cancer Lixia Xu, Xiaoxing Li, Eagle SH Chu, Guijun Zhao, Minnie Y. Go, Zhirong Zeng, Qian Tao, Joseph J. Sung, Jun Yu Background & Aims: Using methylation sensitive arbitrary primed PCR, we identified that B cell CLL/lymphoma 6 member B (BCL6B) gene was preferentially methylated in cancer. In this study, the epigenetic regulation, biological function and clinical significance of BCL6B were examined in gastric cancer (GC). Materials & Methods: BCL6B promoter methylation was evaluated by methylation specific PCR, combined bisulfite restriction analysis and bisulfite genomic sequencing in 9 GC cell lines and in two cohorts of 208 and 101 GC patients from two single institutions, as well as in 20 normal controls. Immunohistochemistry was performed to assess the protein expression of BCL6B in 12 paired GC tissues. The biological functions of BCL6B in GC cell lines were determined by cell viability assay, colony formation and cell cycle analyses, as well as In Vivo tumorigenicity assay. Molecular targets of BCL6B were identified by cDNA expression array. Log-rank test and Cox regression were used to identify independent predictors of prognosis. Results: BCL6B was inactivated in all (9/9) GC cell lines, but was readily expressed in normal gastric tissues. The protein expression of BCL6B was significantly lower in primary GCs as compared to their adjacent normal tissues (P<0.001). The loss of BCL6B gene expression was revealed to be regulated by promoter hypermethylation. The BCL6B expression in silenced GC cell lines could be restored by demethylation agent, 5-aza-deoxycytidine. Ectopic re-expression of BCL6B by stable transfection of BCL6B in AGS and BGC823 cells inhibited colony formation (P<0.001 for both cell lines), suppressed cell viability (P<0.001 for both cell lines) and induced apoptosis (P<0.001 for both cell lines). The In Vivo growth of BGC823 cells in nude mice was also markedly inhibited after stable expression of BCL6B (P<0.001). The anti-tumorigenic function of BCL6B was regulated by up-regulating tumor suppressors ATM and p53. Induction of apoptosis was mediated by enhanced cleaved caspase-3, 7, 8, 9 and PARP, suggesting BCL6B induced apoptosis through both intrinsic and extrinsic caspase dependent pathways. BCL6B methylation was detected in 49.0% (102/208) and 66.3% (67/101) of two cohort GC patients, but was not detected in 20 normal gastric tissues. Multivariate analysis showed that BCL6B methylation is an independent factor to the survival of GC patients (P=0.001 for cohort I and P=0.055 for cohort II). Kaplan-Meier survival curves showed that these methylated patients had significantly shorter survival in the both cohorts (P<0.05, log-rank test). Conclusion: Epigenetic inactivation of BCL6B may play a pivotal role as a potential tumor suppressor in gastric carcinogenesis. Detection of methylated BCL6B may serve as an independent biomarker for the prognosis of GC. Acknowledgment The project was supported by Research Grant ERG CUHK (GRF 473008).
952 Deregulated Hedgehog Signaling in the Pathogenesis of Barrett's Disease and Gastric Adenocarcinoma Alexandre Ermilov, Jennifer Ferris, Joanna Pero, Xiaotan T. Qiao, Joel K. Greenson, Deborah L. Gumucio, Andrzej Dlugosz Deregulated activation of the Hedgehog (Hh) signaling pathway plays a central role in the development of basal cell carcinoma in skin and has also been implicated in the pathogenesis of several epithelial neoplasms arising in the gastrointestinal tract, including gastric adenocarcinoma, the second leading cause of cancer mortality worldwide. However, a causal role for Hh signaling in the pathogenesis of these tumors has not been established, and there is controversy regarding which cell types respond to physiologic Hh signaling in normal stomach. We show that mice engineered to mimic oncogenic Hh signaling in stomach, using an activated form of the transcription factor GLI2 (GLI2*), develop invasive gastric adenocarcinomas which frequently arise near the squamocolumnar junction (SCJ). To identify Hh-responsive potential tumor progenitor cells in this region and elsewhere in stomach, we mapped Hh pathway activity using Gli1-, Gli2-, and Ptch1-lacZ reporter mice. In corpus and antrum, Hh-responsive cells were restricted to mesenchymal cell populations, which included myofibroblasts and smooth muscle cells, in keeping with the pattern of Hh signaling in the intestine. In most of the forestomach, Hh signaling was also limited to the mesenchyme; however, a subset of Hh-responsive basal epithelial cells was identified in the forestomach limiting ridge at the SCJ. To study the consequences of uncontrolled Hh pathway activation in forestomach epithelium, we generated bitransgenic mice carrying: a) a Cre-inducible GLI2* transgene (CLEG2) under the control of a ubiquitous promoter and b) a K5-Cre transgene which drives recombination in forestomach. K5-Cre;CLEG2 mice, in which GLI2* expression was activated in forestomach squamous epithelium, developed a dysplastic, Barrett's-like epithelium near the SCJ. While most of the abnormal cells expressed keratins K8 and K19 (markers of glandular epithelia), a subset of cells co-expressed keratin K5 or K14 (squamous epithelium markers), supporting the idea that the dysplastic Barrett's-like lesions are derived from Hh-responsive basal cells in the forestomach. A similar pattern of glandular and squamous keratin co-expression was also seen in early mouse gastric adenocarcinomas arising near the SCJ. Our data point to a causal role for deregulated Hh signaling in the pathogenesis of Barrett's disease and gastric adenocarcinoma, and identify Hh-responsive forestomach basal cells near the SCJ as the likely progenitor cells that are reprogrammed to give rise to these glandular lesions.
950 DACT1 is Silenced by CpG Methylation in Gastric Cancer and Contributes to the Pathogenesis of Gastric Cancer Shiyan Wang, Minnie Y. Go, Eagle SH Chu, Kin-Fai Cheung, Xiaoxing Li, Wei Kang, Joanna H. Tong, Qian Tao, Ka Fai To, Joseph J. Sung, Jun Yu Background and Aims: DACT1 is a Dishevelled (DVL) antagonist in planar cell polarity (PCP) and noncanonical WNT signaling. Using methylation sensitive arbitrary primed PCR and RT-PCR, DACT1 was preferentially methylated and downregulated in gastric cancer. We aim to clarify its epigenetic inactivation, biological function and clinical significance in gastric cancer. Materials and Methods: Methylation status was evaluated by MSP and direct bisulfite sequencing (BGS) in 20 gastric cancer cell lines and 205 primary gastric cancers. The effects of DACT1α re-expression were determined in cell growth, apoptosis, proliferation, spreading, migration and invasion assays. Luciferase reporter assays were performed to evaluate DACT1α downstream cellular signaling pathways. DACT1α target genes were identified by Cancer Pathway complementary DNA microarray analysis. Results: DACT1 was silenced or downregulated in 50% (10/20) of gastric cancer cell lines, but was expressed in normal gastric tissues. MSP and direct BGS results indicated that promoter methylation was a major mechanism for the transcriptional silencing of DACT1 in gastric cancer cells. Ectopic expression of DACT1α in silenced gastric cancer cell lines (AGS, BGC823 and MGC803) by stable transfection of DACT1α suppressed colony formation (P<0.001 for all three cell lines). Overexpression of DACT1α induced apoptosis in BGC823 (P=0.031) and in MGC803 (P=0.004). Induction of apoptosis was mediated by activating caspase 3, caspase 7, caspase 9 and PARP. In addition, DACT1α inhibited cell migration both in AGS (P<0.001) and in MGC803 (P=0.001), suppressed cell invasion in AGS (P=0.006) and in MGC803 (P= 0.001) and dampened cell adhesion spreading ability along with reduced actin microfilament (stress fiber) formation. Moreover, DACT1α inhibited AP-1 and NF-κB signalings and decreased the expression of NF-κB signaling mediator NFKB1 (p50) and its downstream oncogenic factors IL8 and TNFα. DACT1α also led to downregulation of angiogenic PDGFB, VEGFA and TGFB1, oncogene ETS2 as well as multiple cell migration and invasion molecules ITGA1, ITGA2, ITGA3, ITGB3, MCAM, MMP9 and PLAU. DACT1 methylation was detected in 29.3% (60/205) of primary gastric tumors, but not in 20 normal gastric tissues. DACT1 methylation was significantly associated with advanced tumor size grade (P=0.013), lymph
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AGA Abstracts
AGA Abstracts
expression of cytoskeletal markers of quiescent stellate cells. Gene-expression profiling identified IL-8 signalling and the wnt-β-catenin pathway as the key canonical pathways affected. Organotypic co-culture of PSCs and cancer cells demonstrated an indirect effect of treated PSCs on cancer cell morphology, proliferation (decrease) and apoptosis (increase) and confirmed the engagement of the wnt-β-catenin signalling pathway. There was less βcatenin signalling (TOPFlash reporter assays) in cancer cells co-cultured with treated quiescent PSCs, which was mediated by an increased expression of sFRP4 (secreted frizzledrelated protein 4) resulting in decreased invasion of cancer cells. Additionally, in contrast to vehicle treated orgnaotypic cultures, PSCs exposed to ATRA did not invade into the extracellular matrix gel but formed a “wall” at the cancer-matrix junction, blocking cancer cell invasion. Conclusion: Targeting the desmoplastic stroma with agents such as ATRA interferes with the, for tumour progression important, tumour-stroma cross-talk and offers exciting opportunities for combinatorial therapy for pancreatic cancer.
949 Epigenetic Inactivation of BCL6B, a Functional Tumor Suppressor for Gastric Cancer, is Associated With Poor Survival of Gastric Cancer Lixia Xu, Xiaoxing Li, Eagle SH Chu, Guijun Zhao, Minnie Y. Go, Zhirong Zeng, Qian Tao, Joseph J. Sung, Jun Yu Background & Aims: Using methylation sensitive arbitrary primed PCR, we identified that B cell CLL/lymphoma 6 member B (BCL6B) gene was preferentially methylated in cancer. In this study, the epigenetic regulation, biological function and clinical significance of BCL6B were examined in gastric cancer (GC). Materials & Methods: BCL6B promoter methylation was evaluated by methylation specific PCR, combined bisulfite restriction analysis and bisulfite genomic sequencing in 9 GC cell lines and in two cohorts of 208 and 101 GC patients from two single institutions, as well as in 20 normal controls. Immunohistochemistry was performed to assess the protein expression of BCL6B in 12 paired GC tissues. The biological functions of BCL6B in GC cell lines were determined by cell viability assay, colony formation and cell cycle analyses, as well as In Vivo tumorigenicity assay. Molecular targets of BCL6B were identified by cDNA expression array. Log-rank test and Cox regression were used to identify independent predictors of prognosis. Results: BCL6B was inactivated in all (9/9) GC cell lines, but was readily expressed in normal gastric tissues. The protein expression of BCL6B was significantly lower in primary GCs as compared to their adjacent normal tissues (P<0.001). The loss of BCL6B gene expression was revealed to be regulated by promoter hypermethylation. The BCL6B expression in silenced GC cell lines could be restored by demethylation agent, 5-aza-deoxycytidine. Ectopic re-expression of BCL6B by stable transfection of BCL6B in AGS and BGC823 cells inhibited colony formation (P<0.001 for both cell lines), suppressed cell viability (P<0.001 for both cell lines) and induced apoptosis (P<0.001 for both cell lines). The In Vivo growth of BGC823 cells in nude mice was also markedly inhibited after stable expression of BCL6B (P<0.001). The anti-tumorigenic function of BCL6B was regulated by up-regulating tumor suppressors ATM and p53. Induction of apoptosis was mediated by enhanced cleaved caspase-3, 7, 8, 9 and PARP, suggesting BCL6B induced apoptosis through both intrinsic and extrinsic caspase dependent pathways. BCL6B methylation was detected in 49.0% (102/208) and 66.3% (67/101) of two cohort GC patients, but was not detected in 20 normal gastric tissues. Multivariate analysis showed that BCL6B methylation is an independent factor to the survival of GC patients (P=0.001 for cohort I and P=0.055 for cohort II). Kaplan-Meier survival curves showed that these methylated patients had significantly shorter survival in the both cohorts (P<0.05, log-rank test). Conclusion: Epigenetic inactivation of BCL6B may play a pivotal role as a potential tumor suppressor in gastric carcinogenesis. Detection of methylated BCL6B may serve as an independent biomarker for the prognosis of GC. Acknowledgment The project was supported by Research Grant ERG CUHK (GRF 473008).
952 Deregulated Hedgehog Signaling in the Pathogenesis of Barrett's Disease and Gastric Adenocarcinoma Alexandre Ermilov, Jennifer Ferris, Joanna Pero, Xiaotan T. Qiao, Joel K. Greenson, Deborah L. Gumucio, Andrzej Dlugosz Deregulated activation of the Hedgehog (Hh) signaling pathway plays a central role in the development of basal cell carcinoma in skin and has also been implicated in the pathogenesis of several epithelial neoplasms arising in the gastrointestinal tract, including gastric adenocarcinoma, the second leading cause of cancer mortality worldwide. However, a causal role for Hh signaling in the pathogenesis of these tumors has not been established, and there is controversy regarding which cell types respond to physiologic Hh signaling in normal stomach. We show that mice engineered to mimic oncogenic Hh signaling in stomach, using an activated form of the transcription factor GLI2 (GLI2*), develop invasive gastric adenocarcinomas which frequently arise near the squamocolumnar junction (SCJ). To identify Hh-responsive potential tumor progenitor cells in this region and elsewhere in stomach, we mapped Hh pathway activity using Gli1-, Gli2-, and Ptch1-lacZ reporter mice. In corpus and antrum, Hh-responsive cells were restricted to mesenchymal cell populations, which included myofibroblasts and smooth muscle cells, in keeping with the pattern of Hh signaling in the intestine. In most of the forestomach, Hh signaling was also limited to the mesenchyme; however, a subset of Hh-responsive basal epithelial cells was identified in the forestomach limiting ridge at the SCJ. To study the consequences of uncontrolled Hh pathway activation in forestomach epithelium, we generated bitransgenic mice carrying: a) a Cre-inducible GLI2* transgene (CLEG2) under the control of a ubiquitous promoter and b) a K5-Cre transgene which drives recombination in forestomach. K5-Cre;CLEG2 mice, in which GLI2* expression was activated in forestomach squamous epithelium, developed a dysplastic, Barrett's-like epithelium near the SCJ. While most of the abnormal cells expressed keratins K8 and K19 (markers of glandular epithelia), a subset of cells co-expressed keratin K5 or K14 (squamous epithelium markers), supporting the idea that the dysplastic Barrett's-like lesions are derived from Hh-responsive basal cells in the forestomach. A similar pattern of glandular and squamous keratin co-expression was also seen in early mouse gastric adenocarcinomas arising near the SCJ. Our data point to a causal role for deregulated Hh signaling in the pathogenesis of Barrett's disease and gastric adenocarcinoma, and identify Hh-responsive forestomach basal cells near the SCJ as the likely progenitor cells that are reprogrammed to give rise to these glandular lesions.
950 DACT1 is Silenced by CpG Methylation in Gastric Cancer and Contributes to the Pathogenesis of Gastric Cancer Shiyan Wang, Minnie Y. Go, Eagle SH Chu, Kin-Fai Cheung, Xiaoxing Li, Wei Kang, Joanna H. Tong, Qian Tao, Ka Fai To, Joseph J. Sung, Jun Yu Background and Aims: DACT1 is a Dishevelled (DVL) antagonist in planar cell polarity (PCP) and noncanonical WNT signaling. Using methylation sensitive arbitrary primed PCR and RT-PCR, DACT1 was preferentially methylated and downregulated in gastric cancer. We aim to clarify its epigenetic inactivation, biological function and clinical significance in gastric cancer. Materials and Methods: Methylation status was evaluated by MSP and direct bisulfite sequencing (BGS) in 20 gastric cancer cell lines and 205 primary gastric cancers. The effects of DACT1α re-expression were determined in cell growth, apoptosis, proliferation, spreading, migration and invasion assays. Luciferase reporter assays were performed to evaluate DACT1α downstream cellular signaling pathways. DACT1α target genes were identified by Cancer Pathway complementary DNA microarray analysis. Results: DACT1 was silenced or downregulated in 50% (10/20) of gastric cancer cell lines, but was expressed in normal gastric tissues. MSP and direct BGS results indicated that promoter methylation was a major mechanism for the transcriptional silencing of DACT1 in gastric cancer cells. Ectopic expression of DACT1α in silenced gastric cancer cell lines (AGS, BGC823 and MGC803) by stable transfection of DACT1α suppressed colony formation (P<0.001 for all three cell lines). Overexpression of DACT1α induced apoptosis in BGC823 (P=0.031) and in MGC803 (P=0.004). Induction of apoptosis was mediated by activating caspase 3, caspase 7, caspase 9 and PARP. In addition, DACT1α inhibited cell migration both in AGS (P<0.001) and in MGC803 (P=0.001), suppressed cell invasion in AGS (P=0.006) and in MGC803 (P= 0.001) and dampened cell adhesion spreading ability along with reduced actin microfilament (stress fiber) formation. Moreover, DACT1α inhibited AP-1 and NF-κB signalings and decreased the expression of NF-κB signaling mediator NFKB1 (p50) and its downstream oncogenic factors IL8 and TNFα. DACT1α also led to downregulation of angiogenic PDGFB, VEGFA and TGFB1, oncogene ETS2 as well as multiple cell migration and invasion molecules ITGA1, ITGA2, ITGA3, ITGB3, MCAM, MMP9 and PLAU. DACT1 methylation was detected in 29.3% (60/205) of primary gastric tumors, but not in 20 normal gastric tissues. DACT1 methylation was significantly associated with advanced tumor size grade (P=0.013), lymph
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AGA Abstracts
AGA Abstracts
expression of cytoskeletal markers of quiescent stellate cells. Gene-expression profiling identified IL-8 signalling and the wnt-β-catenin pathway as the key canonical pathways affected. Organotypic co-culture of PSCs and cancer cells demonstrated an indirect effect of treated PSCs on cancer cell morphology, proliferation (decrease) and apoptosis (increase) and confirmed the engagement of the wnt-β-catenin signalling pathway. There was less βcatenin signalling (TOPFlash reporter assays) in cancer cells co-cultured with treated quiescent PSCs, which was mediated by an increased expression of sFRP4 (secreted frizzledrelated protein 4) resulting in decreased invasion of cancer cells. Additionally, in contrast to vehicle treated orgnaotypic cultures, PSCs exposed to ATRA did not invade into the extracellular matrix gel but formed a “wall” at the cancer-matrix junction, blocking cancer cell invasion. Conclusion: Targeting the desmoplastic stroma with agents such as ATRA interferes with the, for tumour progression important, tumour-stroma cross-talk and offers exciting opportunities for combinatorial therapy for pancreatic cancer.