M952

Gastrin Stabilizes ~-Catenin Protein Levels in Colorectal Cancer Diane H. Song, Jennifer Kaufman, M. M. Wolfe

Introduction and Purpose: In addition to its recograzed role in the physiological regulation of acid secretion, another biological property attributed to gastrin (G) is its trophic effect on gastrointestinal (G1) mucosa. Numerous studies have demonstrated that G stimulates not only the growth of normal GI epithelial cells, but also malignancies of colorectal (CRC), gastric, and pancreatic etiology. However, the precise mechanisms mediating these trophic effects are unclear. Recent studies have indicated that G is a target of [3-catenin dependent T-cell factor (TCF) transcription. [3-catenin, which serves as a critical co-activator of the Wnt signaling pathway, has previously been shown to activate TCFAymphocyte enhancer binding factor (TCF/LEF) mediated transcription of oncogenes in CRC cells. Methods and Results: To determine the effect of G on various Wnt signaling components, we initially investigated the effect of G on [3-catenin, the necessary co-factor for TCF/LEF dependent transcription. Western analysis of MC-26 cells, a mouse CRC cell line that possesses G receptors, demonstrated induction of [3-cateinn (3-fold at 20 nM), cyclin D1 (2-fold at 50 nM), and COX-2 (2-fold at 10 nM) expression within a period of 4 h in the presence of amidated G-17. In response to the co-incubation of G-17 and L365,260, a G-specific receptor antagonist, the upregufation of [3-eatenin was attenuated. In addition, G-17 augmented LEF- 1 dependent transcription by -80% at 50 nM. Not only did G upregnlate LEF-1 dependent transcription and one of its target proteins, cyclin D1, but it also enhanced promoter activity of a full-length (-1745) cyclin D1 promoter compared to the minimal promoter (-66). Furthermore, half-life analysis of endogenous [3-catenin suggested that the addition of G in the presence of cycloheximide (CHX) prolonged the protein levels of [3-catenin (T~/~ > 6 h) when compared to CHX only (T~/2 -3 h), suggesting that stabilizes [3-catenin protein. Interestingly, CK2 kinase activity, which has been previously reported to phosphorylate [3- eatenin and stabilize its protein levels, was enhanced nearly 2-fold at 20 nM when compared to untreated samples. Summary: The results of these studies suggest that G may promote proliferation in CRC, at least in part, through the stabilization of [3-catenin, possibly via the upregulation of CK2 activity. Conclusion: Our findings are consistent with a novel mechanism that links this important regulatory peptide with factors that enhance oncogenic 13-catenin protein and its central role in tumorigenesis.

M953

Induction of E-cadherin Through SNAIL Downregulation: Potential Mechanism for Suppression of ~-catenin Signaling by Sulindac Sulfide in HCT-116 Cells Hemam K. Roy, Jennifer L. Koetsier, Thomas A. Victor, Ramesh K. Wall

Inhibition of 13-catenin signahng is important in the prevention of colorectal cancer (CRC) by NSAIDS. Both in vivo and in vitro data suggest that NSAIDS can decrease nuclear [3- catenin, and hence transcriptional activity, without altering total cellular [3-eatenin levels. [3-catenin is avidly bound to the plasma membrane by the cell-adhesion protein E-cadherin. The frequent E-eadherin loss during colon carcinogenesis (known to promote both initiation and progression of CRC) may, therefore, increase nuclear [3-catenin. While the mechanisms for E-cadherin loss in CRC has not been explored, overexpression of the transcriptional repressor SNAIL has been implicated in a number of malignancies. We, therefore, hypothe- sized that SNAIL is important in colon cancer, and that NSAIDS may target the SNAIL/E- cadherin axis. Methods: After reaching 40-50% confluence, HCT-116 cells (a microsatelhte unstable human colon cancer line) were treated with the NSAID sulindac sulfide (SS) or vehicle. Western blot analysis of cell lines and immunohistochemistry (IHC) in colon tumors were performed using standard techniques. Results: SS treatment of HCT-116 cells failed to decrease cellular [3-catenin mass but did suppress nuclear localization. SS caused a dose- dependent decrease in SNAIL with a concomitant increase in E-cadherin (figure 1). This response was time-dependent with maximal effect (seen at day 4) characterized by a 4-fold reduction in SNAIL and 2-fold induction in E-cadherin. IHC revealed heterogeneous nuclear expression of SNAIL in >50% of human and experimental CRC. Conclusions: We demon- strate, for the first time, that NSAIDS inhibit SNAIL expression, resulting in E-cadherin upregulation. Moreover, this is the first report of SNAIL upregnlation in CRC. These novel findings provide insights into CRC biology and the mechanisms of action of NSA1D in CRC chemoprevention.

FIGURE 1

200

�9 "6" II SNAIL

100 l II E-cAD''tERIN 50

0

50 75 100

$ullndac sulfide (micromolar)

M954

Conjugated Linoleic Acid (CLA) Induces Apoptosis and Inhibits Coupling of Heregulin (HRG) Signaling to Phosphatidylinositol 3-Kinase (PI3K) and Akt Via ErbB3 Phosphorylation in Human Colon Cancer Ht-29 Cells Jung H. Park, Han J. Cho, Woo K. Kim, Eun J. Kim, Angela L. Tyner

The incidence of colorectal cancer is increasing, and there is increasing urgency to develop strategies to prevent this disease. With regard to prevention, diet has drawn considerable attention in recent years. CLA is a mixture of positional and geometric isomers of octadecadie- noic acid with conjugated double bonds. CLA has chemoprotective properties in experimental cancer models and in vitro studies have shown that CLA inhibits HT-29 colon cancer cell growth. Members of the ErbB receptor family, including epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4 frequently contribute to the development of several human cancers. To examine CLA regulation of HT-29 cell proliferation and apoptosis and the influence of CLA on the ErbB3 signaling pathway, HT-29 cells were cultured in the presence of CLA and/or the ErbB3 ligand heregulin. CLA inhibited DNA synthesis and induced apoptosis of HT-29 cells. Heregulin-A increased cell growth but did not prevent CLA-induced growth inhibition. EGFR, ErbB2, and ErbB3 were detected in HT-29 cells, and CLA decreased the levels of these proteins in a dose-dependent manner. Immunoprecipi- tation/Westem blot studies revealed that CLA inhibited heregnlin-A-stimulated phosphoryla- tion of ErbB2 and ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) to the ErbB3 receptor, ErbB3-associated PI3K activities, and phospborylation of Akt. HT-29 cells also produced heregnlin, but CLA did not affect the levels of this protein. In conclusion, we have shown that CLA inhibits cell proliferation and stimulates apoptosis in HT-29 cells, and that this may be mediated by its ability to downregnlate ErbB3 signaling and the PI3K/Akt pathway. This study was supported by a grant of the Korea Health 21 R & D Project, Ministry of Heath & Welfare, Republic of Korea (02-PJ1-PG 10-22003-0001 ).

M955

Phosphorylation of CDX-2 at Serine 60 by Mitogenic Signaling Suppresses Guanylyl Cyclase C Expression in Human Colon Carcinoma Cells Matthew D. P. Di Guglielmo, Edmond H. H. Rings, Francois Boudreau, Shiva Kazerounian, lnez A. Ruiz-Stewart, Giovanni M. Pitari, Stephanie Schulz, Scott A. Waldman

Guanylyl cyclase C (GC-C), expressed specifically by intestinal epithelial cells, inhibits the proliferation of those cells upon activation by specific ligands such as the E. coil heat-stable enterotoxin (ST). Enterocyte-specific expression of GC-C is regulated by the transcription factor CDX-2. Of significance, phosphoryLation of CDX-2 at serine 60 "(P-CDX-2-S60) by MAP kinase kinase (MEK) reduces its transactivating capacity. CDX-2 is phosphorylated at serine 60 (P-CDX-2-S60) along a gradient that is high in the crypt and absent in villus enterocytes, in contrast to GC-C expression, which is low in the crypt and high in the villus. Thus, GC-C expression may be regulated by the gradient of P-CDX-2-S60 along the crypt- villus axis. The present study examined the regulation of GC-C expression by phosphorylation of CDX-2 at serine 60 in human colon carcinoma cells in vitro. Transactivation of a GC- C promoter-luciferase construct was increased by co-expression with a phosphorylation- incompetent CDX-2 mutant in which serine 60 was replaced with alainne. Pborbol ester (PMA), which activates mitogenic signaling pathways, induced the phosphorylation of CDX- 2 at serine 60 and inhibited transactivation of the GC-C promoter-luciferase construct in colon carcinoma cells, and expression of GC-C mRNA and GC-C protein, reflected in decreased ST receptor binding sites and ST-stimulated cGMP accumulation in those cells. CDX-2 phosphorylation and the associated reduction in GC-C promoter activity induced by PMA were mediated by a calcium-independent isoform of protein kinase C (PKC) and MEK. In conclusion, mitogenic signaling induced by PMA promoted CDX-2 phosphorylation at serine 60 by a PKC- and MEK-dependent mechanism, which reciprocally suppressed GC- C expression and its antiprollferative effects, supporting the suggestion that GC-C expression is regulated by the gradient of P-CDX-2-S60 along the crypt-villns axis

M956

No-Donating Aspirin Modulates MAP Kinase Signaling in Human Colon Cancer Cells: Implications for Its Growth Inhibitory Effect Tomas Hundley, Jennifer Harley, Edward Lebovics, Basil Rigas

INTRODUCTION: NO-NSAIDs, consisting of a traditional NSA1D to which a NO-donating group is covalently attached via a spacer molecule, are emerging as a safe and effective group of compounds. We have demonstrated that NO-aspirin (NO-ASA) is highly potent in inhibiting the growth of colon and other cancer cell lines by inhibiting cell proliferation and enhancing cell death (Cancer Res 61:3285; J Pharm Exp Ther, 303: 1273). NO-ASA is chemopreventive against colon cancer in animal tumor models. Here we examined whether NO-ASA acts by modulating cell signaling pathways affecting cell kinetics. METHODS: We studied HT-29 and SW-480 colon cancer cells treated with various concentrations of NO- ASA or ASK Cell numbers and viability (trypan blue) were determined. Proteins of the MAP kinase family were analyzed by Western Blotting and densitometry. RESULTS: NO-ASA inhibited the growth of both HT-29 and SW-480 cells (IC50 = 5-8 uM and 50 uM, respectively); the IC50 for ASA was >5 mM. After treatment with NO-ASA, several members of the MAP kinase family were activated in a time- and concentration- dependent manner. For example at one hour, 10 uM NO-ASA induced the following changes: (a) Extracellular signal-regulated kinase 1 and 2 (ERK1/2): three-fold increase in phosphorylation. (b) Akt: two-fold increase in phosphorylation. (c) c-Jun N-terminal kinase (INK): nine-fold increase in phosphorylation. Similar effects were seen with the SW-480 cellline. In contrast, traditional ASA (0.1 raM, 1.0 mM and 5.0 mM) decreased phosphorylation of Erkl/2, Akt, and JNK, compared to control. CONCLUSIONS: NO-ASA markedly stimulates the ERKI/2, Akt, and JNK cell signaling pathways. These changes may alter cell proliferation and cell death thus mediating the growth inhibitory effect of NO-ASK Furthermore, traditional ASA at mM concentrations failed to induce such changes. The difference in these responses between

A-281 AGA Abstracts