Microbiota-gut-brain axis: enteroendocrine cells and the ...
256 Unanticipated Effects of Notch Activation in Enteroendocrine Precursor Cells on Secretory...
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AGA Abstracts 254 TRIM44: From Prognosis to Therapy in Esophageal Adenocarcinoma and Breast Cancer Chin-Ann Johnny Ong, Nicholas Shannon, Pierre Lao-Sirieix, Caryn Ross-Innes, Maria O'Donovan, Oscar M. Rueda, Elaine C. Walker, Ayesha Noorani, Richard H. Hardwick, Carlos Caldas, Rebecca Fitzgerald INTRODUCTION The incidence of esophageal adenocarcinoma (EA) has quadrupled in the last 30 years and outcomes remain poor. Unlike other epithelial cancers, targeted therapies for EA are at an early stage. Using gene expression profiling, we have previously identified TRIM44 as an independent prognostic gene in EA. TRIM proteins are involved in many biological processes and are increasingly recognised to be involved in oncogenesis of various malignancies. AIMS The aims of this project were to 1)Explore the mechanism of dysregul- ation of TRIM44 and association with prognosis. 2)Examine the oncogenic potential of TRIM44 in EA and other epithelial cancers 3)Identify therapeutic options exploiting TRIM44 dysregulation. METHODS AND RESULTS Analysis of our EA gene expression microarray data (n=75), of a large independent matched aCGH and expression microarray of 997 breast cancers and of an online database (Tumourscape n=1932,various epithelial tumours) revealed focal amplification of TRIM44 in 8% of EA, 6% of breast cancers and 4% of epithelial tumours. Validation of amplification in EA was performed using FISH on tissue microarrays from EA (n=164). Expression of TRIM44 was copy number driven in both EA and breast cancer and as per EA, amplification conferred a poor prognosis in breast cancers (p = 0.037). Functional work demonstrated oncogenic addiction to TRIM44 in cell line models harbouring amplifications. siRNA knockdown in HSC39 and JIMT-1, which harbours amplifications and high expression of TRIM44, decreased proliferation of cells by 2 fold (p <0.05) and increased the subG0 fraction on FACS (2.5 fold, p <0.05). In contrast, siRNA treatment of OE19 (low TRIM44 expression) had no effect on viability and proliferation. Overexpression of TRIM44 in Hela cells using a Tet-inducible system increased proliferation (2.5 fold, p = 0.0038) and invasiveness (2 fold, p < 0.05). Taken together, these results suggest that TRIM44 is a putative oncogene. Analysis of gene expression data (EA and breast) identified a potential link between TRIM44 and the mTOR pathway, and suggested sirolimus (mTOR inhibitor) as a therapeutic option. Following validation of the link between TRIM44 and mTOR pathway in the cell line panel (HSC39, JIMT-1, Hela), cell lines with amplification and high expression of TRIM44 were highly sensitive to mTOR inhibition (IC50 <30nm). CONCLUSION TRIM44 is amplified in >5% of EA leading to increased proliferation and invasion In Vitro. Our data suggest a mode of action of TRIM44 via the mTOR pathway. Evaluation of mTOR inhibitors in EA tumours is worthy of consideration and these are currently being evaluated in phase I/II oncology clinical trials including epithelial cancers (eg. renal cell carcinoma and lung cancer). Assessment of TRIM44 amplification status may allow selection of patients who are more likely to respond. 255 Aurka Regulates Hdm2 E3-Ubiquitin Ligase in Esophageal Adenocarcinoma Cells Vikas Sehdev, Ahmed M. Katsha, Abbes Belkhiri, Wael El-Rifai Background: Esophageal adenocarcinomas (EAC) exhibit intrinsic resistance against chemo- therapy. AURKA regulates cell cycle progression and its overexpression is associated with oncogenic transformation. We have recently reported that AURKA is significantly overex- pressed in about 70% of human EAC tissue samples and EAC cell lines. We have previously shown that AURKA inhibits p53- and p73-mediated apoptotic pathways in GI adenocarcin- omas. HDM2 is an E3-ubiquitin ligase which is closely involved in regulating p53 and p73 protein stability and activity. The phosphorylation of Ser166 site in HDM2 protein enhances HDM2 protein activity and stability. Methods and Results: In this study we demonstrate that AURKA directly interacts with HDM2 and regulates HDM2 protein expression and phosphorylation in both FLO-1 and OE33 EAC cells. Western blot analyses were done following AURKA overexpression with adenovirus, knockdown with si-RNA or inhibition with MLN8237 (0.5μM) in FLO-1 and OE33 EAC cell lines. The data indicated that overexpr- ession of AURKA induced both total and phospho-HDM2-(Ser166) protein levels. Knock- down or inhibition of AURKA significantly decreased expression of both total and phospho- HDM2-(Ser166) protein levels in FLO-1 and OE33 EAC cells. Additionally, following aden- ovirus mediated overexpression of AURKA, co-immunoprecipitaion (Co-IP) was done for AURKA and HDM2 in FLO-1 and OE33 EAC cells. The two-way Co-IP data indicated the presence of HDM2 in a complex associated with AURKA and vice-versa. The data from In Vitro protein kinase assay indicated that recombinant AURKA directly phosphorylates recombinant HDM2 at Ser166 site. To confirm direct interaction between recombinant AURKA and HDM2 proteins we performed IP following the In Vitro kinase assay. The In Vitro kinase IP data indicates that kinase intact recombinant AURKA directly interacts and phosphorylates recombinant HDM2 protein. Conclusions: Our data indicate, for the first time, that AURKA directly interacts with HDM2 and phosphorylates it at Ser166 site. This finding suggests that AURKA-mediated regulation of HDM2 could be a major underlying mechanism in regulating p53- and p73-dependent apoptosis. 256 Unanticipated Effects of Notch Activation in Enteroendocrine Precursor Cells on Secretory Lineage Cell Fates Joyce Li, Archana Kapoor, Andrew B. Leiter Notch activation in multipotential progenitor cells in the intestine appears to select against secretory lineage differentiation. In many other organ systems, Notch signals reiteratively control alternate cell fate decisions at distinct stages of differentiation. It is not known whether Notch directly acts on enteroendocrine cells or their precursors. Our aim was to determine if enteroendocrine precursors respond to Notch signaling. We conditionally activ- ated Notch signaling in early and late enteroendocrine precursors by crossing either Ngn3- cre or NeuroD-cre mice to Rosa-Notch mice that express the intracellular effector domain S-60 AGA Abstracts of Notch1 (NICD) and EGFP following Cre mediated recombination. The resultant coexpres- sion of EGFP with NICD facilitated identification of cells where Notch was activated. Notch activation in Ngn3+ cells resulted in death by postnatal day 3, prior to the maturation of secretory lineages. Therefore, we generated inducible Ngn3-creERT2 mice to enable us to study the effects of Notch in adult mice, when all four intestinal cell types have fully developed. Notch activation in Ngn3+ early precursor cells in neonatal and adult intestinal epithelium did not preclude the specification of endocrine cells, but severely limited enteroen- docrine cell number. We identified individual and groups of adjacent EGFP+ cells that did not stain for the endocrine marker Chromogranin A, implying that they adopted an alternate cell fate. A number of EGFP+ cells expressed the brush border enzymes lactase in neonatal and alkaline phosphatase in adult mice, identifying them as enterocytes. We also found EGFP+ cells that stained for the Goblet cell marker Muc2 throughout intestine. Our previous lineage analysis showed that 13% of goblet cells in duodenum and less than 1% in the colon arose from Ngn3+ cells. Surprisingly we observed an increase in Goblet cell numbers arising from Ngn3+ cells following Notch activation, including a nearly 50% increase in colon, suggesting that Notch favors a Goblet cell fate at this developmental stage. Although our prior studies showed that nearly 40% of Paneth cells arose from Ngn3+ precursors, we were unable to identify any EGFP+ cells that stained for Paneth cell markers, suggesting that Notch activation strongly selected against Paneth cells at this stage of differentiation. Unlike Ngn3+ cells, Notch activation in late enteroendocrine precursor cells expressing NeuroD had no apparent effects on any of the four epithelial cell types. In conclusion, our data suggest that Notch activation in intestinal Ngn3+ cells favors enterocyte and goblet cell fates, while selecting against endocrine and Paneth cell differentiation. In contrast, NeuroD+ cells have lost the ability to respond to Notch. Our results suggest that the effects of Notch on secretory lineage cell fates may depend on timing and context of the Notch signal. 257 Gastric Sonic Hedgehog Acts as a Chemoattractant for Macrophage Recruitment During Tissue Repair Amy C. Engevik, Jason R. Martin, Julie A. Tritschler, Rui Feng, Michael A. Schumacher, Yana Zavros Background: During repair Sonic Hedgehog (Shh) regulates multiple events essential for wound healing in various tissues that include cornea, heart and skin. However, the role of Shh as a regulator of gastric tissue repair remains unknown. Loss of Shh expression in the stomach associated with Helicobacter pylori (H. pylori) infection correlates with reduced ulcer repair and increased risk of cancer. Hypothesis: Macrophages play a central role in tissue repair. The recruitment of macrophages, that express components of the Hedgehog signaling pathway, serves as a potential mechanism through which Shh drives tissue repair. Methods: A mouse model expressing a parietal cell-specific deletion of Shh (PC-Shh KO ) was used. Acetic acid-ulcers were induced in control, PC-Shh KO , and H. pylori infected mice and analyzed 1 to 7 days post-ulcer induction. Gastric samples were collected from the ulcerated and adjacent uninjured tissue, epithelium was enzymatically dissociated and macrophage recruitment was analyzed by fluorescence-activated cell sorting (FACS). Changes in cytokine, chemokine and pro-angiogenic factors were measured using a Luminex® based multiplex assay. Changes in tissue Shh concentrations were measured by ELISA. Results: In controls, ulcers healed within 7 days post-injury. Tissue regeneration was accompanied by the recruit- ment of CD11b + F4/80 + Ly6C high macrophages to the stomach within 48 hours post injury. Control mice had elevated expression of chemokines MCP-1 and MIP-2 and pro-angiogenic factor VEGF that correlated with an increased Shh tissue concentration (22,185 ± 2.49 pg/ ml) within 3 days post-injury. PC-Shh KO mice showed complete loss of ulcer repair, the absence of macrophage recruitment and reduced Shh tissue concentrations (8,875 ± 1.61 pg/ml). PC-Shh KO mice exhibited epithelial hyperproliferation and tumor development 7 days post ulcer-induction. Compared to the controls, H. pylori-infected mice had reduced Shh tissue concentrations (6,455 ± 1.94 pg/ml) that increased in response to injury (13,345 ± 5.87 pg/ml) but were still significantly lower compared to controls. Macrophage gastric influx was not observed until 5 days post-injury that was accompanied by suppressed expression of VEGF expression and impaired gastric repair. H. pylori-infected mice had increased IFN-γ and IL-1β tissue concentrations. To identify the direct role of Shh signaling in macrophage recruitment to the stomach during repair, experiments using mice with a myeloid cell-specific deletion of Hedgehog receptor smoothened are pending. Conclusions: Gastric Shh facilitates tissue repair by acting as a macrophage chemoattractant. H. pylori infection suppresses Shh expression as part of the pathophysiological mechanism that may be detrimental to ulcer healing and a trigger for cancer development. 258 Molecular Underpinnings of Highly Penetrant Duodenal Tumors in Lrig1 Null Mice Yang Wang, Emily J. Poulin, Jeff Franklin, Robert J. Coffey Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) is a negative regulator of the ErbB receptor tyrosine kinases and is dysregulated in a number of malignancies. By lineage tracing and gut irradiation, we have shown that Lrig1 marks largely quiescent stem cells in the intestine. Notably, 14/16 functionally null Lrig1 mice (homozygous Lrig1- CreERT2 mice) develop adenomas in the proximal duodenum by 6 months and these tumors histologically progress over time. The first morphological abnormality observed in these mice is expansion of Brunner's glands at 2 months of age. As early as 3 months, histologically low-grade adenomas are detected overlying the expanded Brunner's glands. There is blurring of the boundary between the two compartments. At the blurred boundary, there is ectopic expression of both the epithelial marker ErbB2 and Brunner's gland marker Tff2, suggesting abnormalities in cell lineage allocation. We also observe increased mRNA expression of and immunoreactivity for the Egfr ligand amphiregulin in both Brunner's glands and adenomas compared to adjacent normal mucosa, as well as elevated Egfr mRNA and protein in the involved tissues, supporting a role for ErbB signaling in tumor formation. In addition, by microarray profiling, we find a marked increase in gastric-specific genes Gkn1, Muc5ac and Tff1, and these findings were confirmed by either RT-PCR and/or immunohistochemistry. These lesions harbor histological changes consistent with gastric metaplasia. Based on these
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TRIM44: From Prognosis to Therapy in Esophageal Adenocarcinoma andBreast CancerChin-Ann Johnny Ong, Nicholas Shannon, Pierre Lao-Sirieix, Caryn Ross-Innes, MariaO'Donovan, Oscar M. Rueda, Elaine C. Walker, Ayesha Noorani, Richard H. Hardwick,Carlos Caldas, Rebecca Fitzgerald
INTRODUCTION The incidence of esophageal adenocarcinoma (EA) has quadrupled in thelast 30 years and outcomes remain poor. Unlike other epithelial cancers, targeted therapiesfor EA are at an early stage. Using gene expression profiling, we have previously identifiedTRIM44 as an independent prognostic gene in EA. TRIM proteins are involved in manybiological processes and are increasingly recognised to be involved in oncogenesis of variousmalignancies. AIMS The aims of this project were to 1)Explore the mechanism of dysregul-ation of TRIM44 and association with prognosis. 2)Examine the oncogenic potential ofTRIM44 in EA and other epithelial cancers 3)Identify therapeutic options exploiting TRIM44dysregulation. METHODS AND RESULTS Analysis of our EA gene expression microarraydata (n=75), of a large independent matched aCGH and expression microarray of 997 breastcancers and of an online database (Tumourscape n=1932,various epithelial tumours) revealedfocal amplification of TRIM44 in 8% of EA, 6% of breast cancers and 4% of epithelialtumours. Validation of amplification in EA was performed using FISH on tissue microarraysfrom EA (n=164). Expression of TRIM44 was copy number driven in both EA and breastcancer and as per EA, amplification conferred a poor prognosis in breast cancers (p = 0.037).Functional work demonstrated oncogenic addiction to TRIM44 in cell line models harbouringamplifications. siRNA knockdown in HSC39 and JIMT-1, which harbours amplificationsand high expression of TRIM44, decreased proliferation of cells by 2 fold (p <0.05) andincreased the subG0 fraction on FACS (2.5 fold, p <0.05). In contrast, siRNA treatment ofOE19 (low TRIM44 expression) had no effect on viability and proliferation. Overexpressionof TRIM44 in Hela cells using a Tet-inducible system increased proliferation (2.5 fold, p =0.0038) and invasiveness (2 fold, p < 0.05). Taken together, these results suggest thatTRIM44 is a putative oncogene. Analysis of gene expression data (EA and breast) identifieda potential link between TRIM44 and the mTOR pathway, and suggested sirolimus (mTORinhibitor) as a therapeutic option. Following validation of the link between TRIM44 andmTOR pathway in the cell line panel (HSC39, JIMT-1, Hela), cell lines with amplificationand high expression of TRIM44 were highly sensitive to mTOR inhibition (IC50 <30nm).CONCLUSION TRIM44 is amplified in >5% of EA leading to increased proliferation andinvasion In Vitro. Our data suggest a mode of action of TRIM44 via the mTOR pathway.Evaluation of mTOR inhibitors in EA tumours is worthy of consideration and these arecurrently being evaluated in phase I/II oncology clinical trials including epithelial cancers(eg. renal cell carcinoma and lung cancer). Assessment of TRIM44 amplification status mayallow selection of patients who are more likely to respond.
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Aurka Regulates Hdm2 E3-Ubiquitin Ligase in Esophageal AdenocarcinomaCellsVikas Sehdev, Ahmed M. Katsha, Abbes Belkhiri, Wael El-Rifai
Background: Esophageal adenocarcinomas (EAC) exhibit intrinsic resistance against chemo-therapy. AURKA regulates cell cycle progression and its overexpression is associated withoncogenic transformation. We have recently reported that AURKA is significantly overex-pressed in about 70% of human EAC tissue samples and EAC cell lines. We have previouslyshown that AURKA inhibits p53- and p73-mediated apoptotic pathways in GI adenocarcin-omas. HDM2 is an E3-ubiquitin ligase which is closely involved in regulating p53 and p73protein stability and activity. The phosphorylation of Ser166 site in HDM2 protein enhancesHDM2 protein activity and stability. Methods and Results: In this study we demonstratethat AURKA directly interacts with HDM2 and regulates HDM2 protein expression andphosphorylation in both FLO-1 and OE33 EAC cells. Western blot analyses were donefollowing AURKA overexpression with adenovirus, knockdown with si-RNA or inhibitionwith MLN8237 (0.5μM) in FLO-1 and OE33 EAC cell lines. The data indicated that overexpr-ession of AURKA induced both total and phospho-HDM2-(Ser166) protein levels. Knock-down or inhibition of AURKA significantly decreased expression of both total and phospho-HDM2-(Ser166) protein levels in FLO-1 and OE33 EAC cells. Additionally, following aden-ovirus mediated overexpression of AURKA, co-immunoprecipitaion (Co-IP) was done forAURKA and HDM2 in FLO-1 and OE33 EAC cells. The two-way Co-IP data indicated thepresence of HDM2 in a complex associated with AURKA and vice-versa. The data fromIn Vitro protein kinase assay indicated that recombinant AURKA directly phosphorylatesrecombinant HDM2 at Ser166 site. To confirm direct interaction between recombinantAURKA and HDM2 proteins we performed IP following the In Vitro kinase assay. The InVitro kinase IP data indicates that kinase intact recombinant AURKA directly interacts andphosphorylates recombinant HDM2 protein. Conclusions: Our data indicate, for the firsttime, that AURKA directly interacts with HDM2 and phosphorylates it at Ser166 site. Thisfinding suggests that AURKA-mediated regulation of HDM2 could be a major underlyingmechanism in regulating p53- and p73-dependent apoptosis.
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Unanticipated Effects of Notch Activation in Enteroendocrine Precursor Cellson Secretory Lineage Cell FatesJoyce Li, Archana Kapoor, Andrew B. Leiter
Notch activation in multipotential progenitor cells in the intestine appears to select againstsecretory lineage differentiation. In many other organ systems, Notch signals reiterativelycontrol alternate cell fate decisions at distinct stages of differentiation. It is not knownwhether Notch directly acts on enteroendocrine cells or their precursors. Our aim was todetermine if enteroendocrine precursors respond to Notch signaling. We conditionally activ-ated Notch signaling in early and late enteroendocrine precursors by crossing either Ngn3-cre or NeuroD-cre mice to Rosa-Notch mice that express the intracellular effector domain
S-60AGA Abstracts
of Notch1 (NICD) and EGFP following Cre mediated recombination. The resultant coexpres-sion of EGFP with NICD facilitated identification of cells where Notch was activated. Notchactivation in Ngn3+ cells resulted in death by postnatal day 3, prior to the maturation ofsecretory lineages. Therefore, we generated inducible Ngn3-creERT2 mice to enable us tostudy the effects of Notch in adult mice, when all four intestinal cell types have fullydeveloped. Notch activation in Ngn3+ early precursor cells in neonatal and adult intestinalepithelium did not preclude the specification of endocrine cells, but severely limited enteroen-docrine cell number. We identified individual and groups of adjacent EGFP+ cells that didnot stain for the endocrine marker Chromogranin A, implying that they adopted an alternatecell fate. A number of EGFP+ cells expressed the brush border enzymes lactase in neonataland alkaline phosphatase in adult mice, identifying them as enterocytes. We also foundEGFP+ cells that stained for the Goblet cell marker Muc2 throughout intestine. Our previouslineage analysis showed that 13% of goblet cells in duodenum and less than 1% in thecolon arose from Ngn3+ cells. Surprisingly we observed an increase in Goblet cell numbersarising from Ngn3+ cells following Notch activation, including a nearly 50% increase incolon, suggesting that Notch favors a Goblet cell fate at this developmental stage. Althoughour prior studies showed that nearly 40% of Paneth cells arose from Ngn3+ precursors, wewere unable to identify any EGFP+ cells that stained for Paneth cell markers, suggestingthat Notch activation strongly selected against Paneth cells at this stage of differentiation.Unlike Ngn3+ cells, Notch activation in late enteroendocrine precursor cells expressingNeuroD had no apparent effects on any of the four epithelial cell types. In conclusion, ourdata suggest that Notch activation in intestinal Ngn3+ cells favors enterocyte and goblet cellfates, while selecting against endocrine and Paneth cell differentiation. In contrast, NeuroD+cells have lost the ability to respond to Notch. Our results suggest that the effects of Notchon secretory lineage cell fates may depend on timing and context of the Notch signal.
257
Gastric Sonic Hedgehog Acts as a Chemoattractant for MacrophageRecruitment During Tissue RepairAmy C. Engevik, Jason R. Martin, Julie A. Tritschler, Rui Feng, Michael A. Schumacher,Yana Zavros
Background: During repair Sonic Hedgehog (Shh) regulates multiple events essential forwound healing in various tissues that include cornea, heart and skin. However, the role ofShh as a regulator of gastric tissue repair remains unknown. Loss of Shh expression in thestomach associated with Helicobacter pylori (H. pylori) infection correlates with reduced ulcerrepair and increased risk of cancer. Hypothesis: Macrophages play a central role in tissuerepair. The recruitment of macrophages, that express components of the Hedgehog signalingpathway, serves as a potential mechanism through which Shh drives tissue repair. Methods:A mouse model expressing a parietal cell-specific deletion of Shh (PC-ShhKO) was used.Acetic acid-ulcers were induced in control, PC-ShhKO, and H. pylori infected mice andanalyzed 1 to 7 days post-ulcer induction. Gastric samples were collected from the ulceratedand adjacent uninjured tissue, epithelium was enzymatically dissociated and macrophagerecruitment was analyzed by fluorescence-activated cell sorting (FACS). Changes in cytokine,chemokine and pro-angiogenic factors were measured using a Luminex® based multiplexassay. Changes in tissue Shh concentrations were measured by ELISA. Results: In controls,ulcers healed within 7 days post-injury. Tissue regeneration was accompanied by the recruit-ment of CD11b+F4/80+Ly6Chigh macrophages to the stomach within 48 hours post injury.Control mice had elevated expression of chemokines MCP-1 and MIP-2 and pro-angiogenicfactor VEGF that correlated with an increased Shh tissue concentration (22,185 ± 2.49 pg/ml) within 3 days post-injury. PC-ShhKO mice showed complete loss of ulcer repair, theabsence of macrophage recruitment and reduced Shh tissue concentrations (8,875 ± 1.61pg/ml). PC-ShhKO mice exhibited epithelial hyperproliferation and tumor development 7days post ulcer-induction. Compared to the controls, H. pylori-infected mice had reducedShh tissue concentrations (6,455 ± 1.94 pg/ml) that increased in response to injury (13,345± 5.87 pg/ml) but were still significantly lower compared to controls. Macrophage gastricinflux was not observed until 5 days post-injury that was accompanied by suppressedexpression of VEGF expression and impaired gastric repair. H. pylori-infected mice hadincreased IFN-γ and IL-1β tissue concentrations. To identify the direct role of Shh signalingin macrophage recruitment to the stomach during repair, experiments using mice with amyeloid cell-specific deletion of Hedgehog receptor smoothened are pending. Conclusions:Gastric Shh facilitates tissue repair by acting as a macrophage chemoattractant. H. pyloriinfection suppresses Shh expression as part of the pathophysiological mechanism that maybe detrimental to ulcer healing and a trigger for cancer development.
258
Molecular Underpinnings of Highly Penetrant Duodenal Tumors in Lrig1 NullMiceYang Wang, Emily J. Poulin, Jeff Franklin, Robert J. Coffey
Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) is a negative regulator ofthe ErbB receptor tyrosine kinases and is dysregulated in a number of malignancies. Bylineage tracing and gut irradiation, we have shown that Lrig1 marks largely quiescent stemcells in the intestine. Notably, 14/16 functionally null Lrig1 mice (homozygous Lrig1-CreERT2 mice) develop adenomas in the proximal duodenum by 6 months and these tumorshistologically progress over time. The first morphological abnormality observed in thesemice is expansion of Brunner's glands at 2 months of age. As early as 3 months, histologicallylow-grade adenomas are detected overlying the expanded Brunner's glands. There is blurringof the boundary between the two compartments. At the blurred boundary, there is ectopicexpression of both the epithelial marker ErbB2 and Brunner's gland marker Tff2, suggestingabnormalities in cell lineage allocation. We also observe increased mRNA expression of andimmunoreactivity for the Egfr ligand amphiregulin in both Brunner's glands and adenomascompared to adjacent normal mucosa, as well as elevated Egfr mRNA and protein in theinvolved tissues, supporting a role for ErbB signaling in tumor formation. In addition, bymicroarray profiling, we find a marked increase in gastric-specific genes Gkn1, Muc5ac andTff1, and these findings were confirmed by either RT-PCR and/or immunohistochemistry.These lesions harbor histological changes consistent with gastric metaplasia. Based on these
