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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.

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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.

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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