187 MicroRNA Expression Profiling of Human Barrett\'s Carcinogenesis

AGA Abstracts

Xenografting of OE33 and FLO1 cells stably expressing GPX7 into nude mice demonstrated no tumor growth or significant reduction in tumor size as compared to controls (P=.001). The OE33 and FLO1 cells stably expressing pcDNA3-GPX7 demonstrated potent antioxidative properties as compared to vector controls. The GPX7-expressing cells generated much less H2O2 upon 150 to 300 uM bile acid (deoxychloric acid, DCA) treatment for 15 to 30 min and displayed less oxidative stress as determined by Flow cytometry of CMH2DCFDA. Furthermore the GPX7 transfected cells displayed less oxidative DNA damage than control cells when exposed to DCA and H2O2, as determined by immunofluorescence analysis of 8-hydroxyguanine (8-OHdG), an oxidative DNA damage marker. Conclusions: These novel results indicate that GPX7 may protect epithelial cells against neoplastic transformation by acting as both a tumor suppressor and an anti-oxidant. Dysfunction of the GPX7 gene in EAC through DNA methylation may contribute to its tumorigenesis.

cell fate decisions during normal as well as disease processes such as intestinal metaplasia in Barrett's esophagus and keratin pearl formation in squamous cell carcinoma. 186 Constitutive Hedgehog Signaling Impairs Maturation and Migration of Esophageal Epithelial Precursor Cells Willemijn A. van Dop, Anja Uhmann, Viljar Jaks, Johan Offerhaus, Marius A. van den Bergh Weerman, Jarom Heijmans, Daniel W. Hommes, James C. Hardwick, Rune Toftgård, Heidi Hahn, Gijs R. van den Brink Morphogens pattern tissues during development and maintain tissue architecture in rapidly renewing adult organs such as the gastrointestinal tract. In the columnar epithelium of the adult intestine we found that hedgehog signaling is exclusively from the epithelium to the mesenchyme where it induces a secondary signal that negatively regulates intestinal precursor cells. The mechanisms of epithelial renewal of the squamous epithelium of the esophagus have remained virtually unexplored. Here we examined the role of Hedgehog signaling in the esophagus of adult mice using conditional mutant mice. To study the effect of Hedgehog on the esophageal epithelium we used two different strategies for conditional activation of Hedgehog signaling in the esophagus. We used tamoxifen inducible Ptch1flox/flox-Rosa26CreERT2 mice resulting in body wide recombination of Ptch1 and mice carrying tetracycline regulated-GLI1 crossed to K5-rtTA mice for inducible expression of GLI1 in skin and esophagus. We examined expression of Sonic Hedgehog, Indian Hedgehog, Ptch1 and Gli1, by quantitative RT-PCR and in situ hybridization. The phenotype of Ptch1 mutant mice was examined using routine histological techniques, transmission electron microscopy and immunohistochemical analysis of cellular proliferation and differentiation markers. Cell migration was examined in BrdU pulsed mice. Precursor cells of the esophagus expressed the Hedgehog receptor Ptch1, indicating that they are direct targets of Hedgehog signaling, in contrast to intestinal precursor cells. Sonic Hedgehog was the main Hedgehog produced in the esophagus. Conditional activation of Hedgehog signaling using two different approaches resulted in the same morphological alterations. We observed palisading of the precursor cells in the basal epithelial layer, and failure of epithelial maturation. In some parts of the epithelial layer small epithelial buds were formed that migrated into the underlying mesenchyme. BrdU labeling studies revealed that precursor cells were retained in the basal layer and failed to migrate to the esophageal lumen. Our results suggest that Hedgehog signaling has different functions throughout the gastrointestinal tract. We previously found that Hedgehog signaling negatively regulates the intestinal epithelial precursor cells in a paracrine manner. Here we describe an autocrine role for Hedgehog signaling in the esophageal epithelium. In the esophagus we find that Hedgehog signaling positively regulates precursor cell fate and retention.

184 Cyclin D1 and EGFR Promote Invasive Growth of Human Esophageal Epithelial Cells in Concert With Impaired Notch Signaling Momo Nakagawa, Shinya Ohashi, Mitsuteru Natsuizaka, Maria E. Vega, Douglas Stairs, Andres J. Klein-Szanto, J. Alan Diehl, Warren S. Pear, Hiroshi Nakagawa, Anil K. Rustgi Introduction: Cyclin D1 and Epidermal growth factor receptor (EGFR) are frequently overexpressed in esophageal squamous cell carcinoma (ESCC) and other squamous cell cancers. Notch signaling is implicated in squamous cell proliferation and differentiation, and may act as a tumor suppressor. While squamous differentiation is maintained in ESCC and its precursor lesions, the role of Notch in esophageal carcinogenesis remains elusive. The role of genetic interactions amongst cyclin D1, EGFR and the Notch signaling pathway were explored in this study. Methods: Immortalized human esophageal cells EPC2-hTERT (keratinocytes) were retrovirally transduced with tetracycline-regulatable (tet-off) cyclin D1 with or without EGFR. Notch was activated by the Notch1 intracellular domain (NICD1) and inhibited by dominant negative mastermind-like1 (DN-MAML1), a genetic pan-Notch inhibitor. Notch activity was assessed by 8xCSL-luciferase reporter assays. Quantitative RTPCR, Western blotting and immunohistochemistry were performed. The squamous epithelium was reconstituted in organotypic (3-D) culture, a form of tissue engineering. Results: NICD1 induced CSL-dependent transcriptional activity and Notch target genes such as involucrin (an early marker of squamous differentiation), potently inhibiting EPC2-hTERT cell proliferation, implying the Notch activity in the cell cycle withdrawal and commitment toward squamous differentiation. In 3-D culture, Cyclin D1 overexpression resulted in basal cell hyperplasia as documented by increased Ki67 labeling index, and that was reversed by doxycycline reducing cyclin D1 to the basal level. Without cyclin D1 overexpression, DNMAML1 did not affect basal cell proliferation, yet severely impaired squamous maturation and terminal differentiation with a loss of filaggrin (a late differentiation marker) in the suprabasal layer. Interestingly, cyclin D1 overexpression antagonized the inhibitory effect of DN-MAML1 upon epithelial stratification while segregating uniquely the proliferative basal cell compartment. EGFR alone did not affect the DN-MAML1 effect. However, when overexpressed along with cyclin D1 and DN-MAML1, EGFR promoted cell invasion in 3D culture. Conclusions: These data indicate that cyclin D1 overexpression may affect Notchmediated squamous differentiation program by preventing cell cycle exit while accumurating an undifferentiated and proliferative cell pool upon Notch inhibiiton. EGFR may facilitate invasion of the Notch inhibited cells with cyclin D1 overexpression, providing a novel mechanistic insight into the interplay amongst key oncogenes and the Notch pathway in esophageal carcinogenesis.

187 MicroRNA Expression Profiling of Human Barrett's Carcinogenesis Matteo Fassan, Stefano Volinia, Jeff Palatini, Marco Pizzi, Raffaele Baffa, Roberto Clemente, Christian Rizzetto, Carlo M. Croce, Giovanni Zaninotto, Ermanno Ancona, Massimo Rugge Barrett's Mucosa (BM) is a metaplastic replacement of the native esophageal (squamous) epithelium (ESq) by columnar-intestinalized mucosa. BM is the main risk factor for Barrett's adenocarcinoma (BAc). MicroRNAs (miRNAs) are a class of small non-coding RNAs that control gene expression by targeting mRNAs; miRNAs deregulation has been associated with Barrett's oncogenesis. To explore the hypothesis that a specific miRNAs signature is associated with BM (and its associated cancer risk) a miRNA microarray analysis (OSU-CCC version 4.0; Ohio State University) compared ESq versus all the spectrum of the phenotypic lesions of the Barrett's carcinogenesis. Specimens were collected at Department of Pathology of Padova University from 14 patients undergone esophagectomy for BAc or High-grade noninvasive neoplasia (HG-NiN). Tissues samples for the microarray study included: ESq= 14 samples; BM= 14; Low-grade non-invasive neoplasia (LG-NiN)= 7; HG-NiN= 5; BAc= 11. The miRNA-profiling study consistently disclosed increased expression of 6 miRNAs (hsamiR-215, hsa-miR-560, hsa-miR-615-3p, hsa-miR-192, hsa-miR-326, hsa-miR-147), and decreased expression of 7 others (hsa-miR-100, hsa-miR-23a, hsa-miR-605, hsa-miR-99a, hsa-miR-205, hsa-let-7c, hsa-miR-203). Microarray results were further validated by qRTPCR, using a different series of 10 consecutive BAc cases (Esq= 10 samples; BM= 6 samples; BAc= 10 samples). Interestingly, some of the miRNAs found deregulated in this series are already known as suitable markers of cancer progression in other models of epithelial oncogenesis (i.e.: hsa-let-7c, hsa-miR-192, hsa-miR-203, hsa-miR-205, hsa-miR-215). The results achieved by the miRNA profiling study were further validated by the immunohistochemical (IHC) analysis on two well-defined miRNA gene targets (p63 [hsa-miR-203] and HMGA2 [hsa-let-7c]). The IHC expression of the protein products was consistent with the corresponding miRNA's deregulation. In particular, p63 immunoreactivity was lost in almost all metaplastic/preneoplastic/neoplastic lesions, whereas HMGA2 expression was up-regulated in HG-NiN and BAc. The achieved results confirm that specific miRNAs are involved in BM carcinogenesis and that they may represent a novel diagnostic/prognostic tool in the characterization of BAc gene targets.

185 NOTCH3 Activation at the Onset of Esophageal Squamous Epithelial Differentiation Shinya Ohashi, Mitsuteru Natsuizaka, Ross A. Kalman, Yizeng Yang, Douglas Stairs, Jonathan P. Katz, Warren S. Pear, Andres J. Klein-Szanto, Hiroshi Nakagawa Introduction: The Notch receptor family regulates cell fate specification and differentiation processes through cell-cell communication. Although Notch has been implicated in squamous cell differentiation and proliferation, its role in esophageal epithelial homeostasis remains to be delineated. Methods: Primary and telomerase-immortalized normal human esophageal keratinocytes were grown in low-calcium serum-free medium. Calcium chloride (Ca2+) was used to induce squamous differentiation in culture. Notch was inhibited by dominant negative mastermind-like1 (DN-MAML), a genetic pan-Notch inhibitor in a regulatable manner (Tet-Off system) or gamma-secretase inhibitors (GSI). 8xCSL-luciferase reporter was transiently transfected to assess Notch activity. Short hairpin RNA was stably transduced by lentivirus to knockdown NOTCH3. Quantitative RT-PCR, Western blotting and immunohistochemistry determined gene expression. The squamous epithelium was reconstituted in organotypic (3-D) culture, a form of tissue engineering. A LoxP flanked conditional DNMAML1f/f transgenic mice were crossed to a mouse expressing Cre recombinase in the esophagus (L2-Cre) to activate DN-MAML1 in a tissue specific fashion. Results: Ca2+ (0.51.8 mM) dose-dependently activated Notch and induced early markers of squamous differentiation such as cytokeratin CK13. Amongst the Notch family members, only NOTCH3 was induced robustly at the mRNA and protein levels. Importantly, NOTCH3 underwent proteolytic cleavage and its intracellular domain was translocated into the nucleus. DNMAML1 and GSIs blocked NOTCH3 activation and CK13 induction along with other Notch target genes such as HES5. Moreover, NOTCH3 knockdown resulted in inhibition of Ca2+mediated CK13 induction. In 3-D culture, DN-MAML1 impaired squamous differentiation as corroborated by a reduced CK13 expression, and that was reversed upon DN-MAML1 shutdown by doxycycline, implying a direct CSL-mediated transcriptional regulation through the canonical Notch pathway. While neither GSIs nor DN-MAML1 affected cell proliferation in 3-D culture using human esophageal cells, DN-MAML1 expression in mouse esophagus culminated in basal cell hyperplasia, suggesting a potential difference in the functional consequences of Notch inhibition between human and mouse. Conclusions: Our innovative approaches imply the Notch signaling in the esophageal epithelial homeostasis. In particular, the role of NOTCH3 in squamous differentiation is novel, and may have implications in

AGA Abstracts

188 High Prevalence of DNA Methylation in Residual Barrett's Esophagus After Endoscopic Ablation Jean Wang, Jennifer Shroff, Mingzhou Guo, Elizabeth A. Montgomery, Hilary Cosby, James Herman, Marcia I. Canto Introduction: Although endoscopic ablation is often successful in eradicating high-grade dysplasia/intramucosal carcinoma in patients with Barrett's esophagus (BE), 23% have residual BE with no dysplasia (ND) or low-grade dysplasia (LGD) after treatment. Although patients who present with BE and ND or LGD at baseline are considered to be at low risk for neoplastic progression, the risk of progression in patients with residual BE (ND or LGD)

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