Smad4 mediated tumor suppression in pancreatic cancer cells through suppression of angiogenesis

AliO AGA ABSTRACTS

GASTROENTEROLOGY Vol. 118, No.4

463

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THE PROTO·ONCOGENE AKT IS OVEREXPRESSED IN HUMAN AND EXPERIMENTAL COLORECTAL CARCINOGENESIS. Bola F. Olusola, Thomas e. Smyrk, Anne Ratashak, Hemant K. Roy, Univ of Nebraska Med Ctr, Omaha, NE.

DETECTION OF JCV DNA SEQUENCES IN COLON CANCER CELL LINES BY FLUORESCENT IN SITU HYBRIDIZATION, Luigi Ricciardiello, Ajay Goel, Dong K. Chang, Christina L. Chang, C. Richard Boland, UC San Diego, La Jolla, CA. We have previously reported the isolation of JCV DNA sequences, a human polyomavirus which encodes for the transforming protein T antigen, in both its episomal and integrated forms from colon cancer samples and the colon cancer cell line SW480 which displays chromosomal instability. However, we were unable to find any of the viral sequences in the microsatellite unstable cell line HCTI16. Polyomaviruses are reported to induce chromosomal instability in cell culture model: T antigen protein can bind to p53 and RB proteins, and furthermore it displays a strong helicase activity which could induce chromosomal breakage by unwinding of double stranded DNA. Aim: To evaluate the presence of JCV DNA in its episomal and/or integrated forms in colorectal cancer cell lines displaying chromosomal or microsatellite instability, in order to determine whether JCV might playa role in determining the transformed phenotype. Methods: Ten colon cancer cell lines were employed in this study: DLDI, HCA7, RKO, HCTl5, HCTlI6, HCTlI6+ch3, LOVO, CACm, SW480, HT29. The human lung fibroblast cell line WI38 was used as negative control. Fish analysis was performed on chromosomal and mitotic cell spreads after collecting cells treated with colcemid which blocks the cells in mitosis. The JCV-Madl strain cloned into a pBR322 vector (pMI-TC) was used as probe. Different biotiny lated probes were generated by nick translation and the biotin-strepdavidin reaction was performed with Cy3 labeled streptavidin following the hybridization. Chromosomes were counterstained with Sytox or DAPI and visualized with a confocal Microscope. Results: In SW480 cells, hybridizations were visible in both chromosomes and cells. The rate of positives was low suggesting that the viral sequences are present in low copy numbers. In the remaining cell lines, no JCV sequences were visualized in either chromosomes or cells. Conclusion: This study confirms the presence of JCV sequences in both integrated and episomal forms, suggesting that JCV could play a role in determining the transformed phenotype.

The inhibition of apoptosis is a crucial early step in the development of colorectal cancer (CRC), although the mechanisms are incompletely understood. In a variety of malignancies (e.g. pancreatic) there is an overexpression of the novel proto-oncogene, Akt (protein kinase B), an antiapoptotic serine-threonine kinase also implicated in Wnt signaling. Since the role of Akt in CRC has not been previously explored, we examined this by immunohistochemistry (lHC) in sporadic human adenomas and CRe. To investigate the microsatellite instability (MSI) pathway, tumors from patients with hereditary nonpolyposis colon cancer (HNPCC) were assessed. Finally, Akt was evaluated in experimental CRC using the azoxymethane (AOM)-rat model. METHODS: IHC was performed on paraffin-embedded samples with a monoclonal and polyclonal antibody for Aktl/2 using the Vectastain ABC kit. Human and animal colon tumors were selected from archived materials. Akt staining of sections was assessed by a pathologist using appropriate negative and positive controls. RESULTS and CONCLUSIONS: As demonstrated by the Table, Akt is overexpressed in approximately 22-42% of human and experimental CRe. The genetic mechanisms of tumor development (adenomatous polyposis coli mutation in sporadic CRC, f3-catenin mutations in AOM-tumors or MSI in HNPCC tumors) did not appear to significantly affect expression of this epigenetic protein. The observation that Akt overexpression occurs early in carcinogenesis (adenomas) emphasizes its biological significance. Our recent demonstration that Akt downregulation is important in nonsteroidal antiinflammatory drugs (NSAID) induction of apoptosis is supported by the observation that AOM-tumors from animals that had been treated with NSAIDS had a trend towards lower Akt expression than those on a control diet (60 versus 28 % although the numbers were small). This data suggests that Akt overexpression is important in the early dysregulation of apoptosis and Wnt signaling which characterizes CRC development.

466 IMMUNOHISTOCHEMICAL STAINING OF TUMORS FOR AKT

Tumors positive Total tumors

ADENOMAS

SPORADIC CRC

HNPCC

AOM·RAT

2(28%) 7

7(30%)

8(22%)

5(42%)

23

36

12

464 INVOLVEMENT OF THE EPIDERMAL GROWTH FACTOR RECEPTOR IN SRC-DEPENDENT ACTIVATION OF EXTRACEL· LULAR-REGULATED KINASES·l AND -2 IN PANCREATIC AR42J CELLS. Albrecht Piiper, Claudio D. Giannini, Robert Elez, Stefan Zeuzem, Dept of Medicine, Univ of Frankfurt, FrankfurtlM., Germany; Dept of Medicine, FrankfurtlM., Germany. Background: In addition to stimulation of exocrine secretion, cholecystokinin (CCK) induces activation of extracellular-regulated kinases-I and -2 (ERK1I2), which play a key role in the regulation of proliferation and differention. Activation of ERKI/2 by G protein-coupled receptors such as the CCK receptor has been described to occur either through a protein kinase C (PKC)-dependent, Ras-independent, or through a pathway involving G if3y-mediated activation of Src family tyrosine kinases (SFTK) and Ras. CCK has been shown to activate ERKI/2 through activation of PKe. Aim: Elucidation of the role of the epidermal growth factor receptor (EGFR) in CCK-induced ERKI/2 activation. Methods: In pancreatic acinar carcinoma AR42J cells, activation of ERK1I2 was investigated by immune-complex kinase assay or Western blotting with antibody recognizing specifically the activated form of ERKII2. Tyrosine phosphorylation of the EGFR and its complex formation with She, Grb2 and Src was examined by immunoprecipitation and Western blotting. Ras activation was studied by binding of activated Ras to recombinant Ras-binding domain of Raf. Ras was inhibited by transient overexpression of dominant-negative Ras. Results: ERK 112activation by CCK was abolished by inhibition of the EGFR (by AGl478), SFTK (by PPl), or expression of dominant-negative Ras; CCK caused activation of Ras, which was inhibited by AG1478. The effects of AGI478 and PPI were specific, because AGl478 did not influence activation of ERKI/2 by hepatocyte growth factor, which activates a receptor tyrosine kinase distinct from the EGFR, and PPI had no effect on ERKII2 activation by EGF. CCK caused tyrosine phosphorylation of the EGFR and She as well as complex formation of the EGFR with She and Grb2; these effects were small as compared to the effect of a maximum effective concentration of EGF itself, indicating that CCK causes low degree EGFR activation. CCK also induced association of Src with the EGFR and She. CCK-induced complex formation between She and Src was inhibited by AG1478. EGF increased the amount of Src in EGFR, but not in She immunoprecipitates. Conclusion: Our results suggest that CCK utilizes the EGFR to assemble the Src-dependent Ras activation complex, which mediates ERKII2 activation. Src appears to be involved in CCK- but not EGF-induced recruitment of the adaptor proteins She and Grb2 to the EGFR.

DPC4/SMAD4 MEDIATED TUMOR SUPPRESSION IN PANCREATIC CANCER CELLS THROUGH SUPPRESSION OF ANGIOGENESIS. Irmgard Schwarte-Waldhoff, Olga V. Volpert, Noel P. Bouck, Bence Sipos, Stephan A. Hahn, Susanne Klein-Scory, Jutta Luettges, Guenter Kloeppel, Ulrich Graeven, Christina Eilert-Micus, Annette Hintelmann, Wolff Schmiegel, Ruhr-Universitaet Bochum, Knappschaftskrankenhaus, Bochum, Germany; Northwestern Univ, Chicago, IL; UniversitSt Kiel, Kiel, Germany; Universitaet Kiel, Kiel, Germany. Background: DPC4/Smad4 is a tumor suppressor gene lost at high frequencies in cancers of the pancreas and other digestive organs. DPC41 Smad4 encodes a key intracellular messenger in the TGF-f3 signaling cascade. Most carcinoma cells are known to have lost sensitivity towards TGF-f3 induced growth inhibition, so the acquisition of TGF-f3 resistance was assumed to be the driving force for loss of DPC4/Smad4 in tumors. However, TGF-f3 effects are manifold and in fact, TGF-f3 cytokines are frequently overexpressed in carcinomas, suggesting that they may also function to promote tumor growth through paracrine mechanisms. Aims: To study mechanisms of DPC4/Smad4 mediated tumor suppression with respect to the dual functions of TGF-f3 cytokines in human tumorigenesis. Results: Although reexpression of DPC4 suppressed tumor formation in vivo it did not restore sensitivity to TGF-f3 in vitro. Rather it decreased expression of vascular endothelial growth factor (VEGF) and increased expression ofthrombospondin-l (TSP-l). These DPC4 mediated shifts in expression levels of angiogenesis mediators VEGF and TSP-l resulted in cells that were anti-angiogenic and produced small non-progressive tumors with reduced vascular density. Conclusions: Our data define the control of an angiogenic switch as a novel mechanism of tumor suppression for DPC4/Smad4 and identify the angiogenic mediators VEGF and TSP-I as key target genes. 467 P63, A P53 HOMOLOGUE, COMPENSATES FOR FUNCTIONAL P53 IN THE ORAL-ESOPHAGEAL EPITHELIUM OF P53 DEFICIENT MICE. Yasir Suliman, Oliver G. Opitz, Anil K. Rustgi, Div of Gastroenterology, Univ of Pennsylvania, Philadelphia, PA. INTRODUCTION: p63, a p53 homologue, has been shown to be critical in normal squamous epithelial development, as well as craniofacial and limb development in men and mice. p63 can bind to p53 responsive promoters and thus activate p53 dependent genes in vitro. but its biological properties in vivo, in particular to p53-like functions have not been explored. Thus, p53 homologues like p63 might compensate for the inactivation of p53 in a tissue specific fashion. METHODS: Immunohistochemistry for full length p63 and the N-terminal truncated transcript of p63, tl.N-p63, was performed. Tongue, esophagus and forestomach of 5 wild type as well as p53 deficient mice were immunostained. Detection was done with the ABC kit. As a functional target of p53 family members, p21 was used in immunostaining in the same group of animals. RESULTS: The p53 homologue, p63, was overexpressed in the oral-esophageal squamous epithe-