In vivo activation of raf-1 inhibits tumor growth and development in a xenograft model of human MTC

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS tion, after intestinal I/R injury. These findings demonstrate that administration of HB-EGF exerts an anti-inflammatory effect during intestinal injury, and further support the use of HB-EGF-based therapeutic strategies in the treatment of injurious intestinal processes including I/R .

252. CO2 PNEUMOPERITONEUM IMPROVES COLITIS IN AN ANIMAL MODEL OF CROHNS DISEASE. A. Aurora 1, J. Fuentes 1, E. Hanly 1, S. Shih 1, A. Demaio 1, M. Talamini 2; 1 Johns Hopkins University, Baltimore, MD, 2University of California, San Diego, San Diego, CA. Introduction: CO2 pneumoperitoneum has the capacity to attenuate the inflammatory response in the acute inflammatory setting. However, the laparoscopic approach is also routinely used when surgical intervention is required in settings of chronic inflammation such as Crohns disease. Because some evidence suggests that minimally invasive approaches to surgery for Crohns disease results in improved outcomes compared to conventional open surgery, we hypothesized that the capnoperitoneum has therapeutic potential in chronic inflammatory settings, such as Crohns colitis. Specifically, we tested whether capnoperitoneum alone without surgical intervention could ameliorate inflammation of the bowel. Methods: Using a well established rodent model of Crohns colitis we induced Crohnslike colitis in rats. Rats were fasted for 48h prior to induction of colitis. Rats were anesthetized for instillation of trinitrobenzene sulfonic acid/ethanol (300ul) into the distal colon using an 8 cm catheter per rectum. The rats were then observed and weighed daily for 8 days with water and food ad libitum. On day 9, all rats were anesthetized for 30 min, half received only anesthesia and half received a CO2 pneumoperitoneum at 4 mmHg. This 30 min procedure was repeated on days 10-12. On day 13, rats were weighed and then sacrificed, the colon harvested and scored by a doubly-blinded observer. Results: Rats in the anesthesia only group had an average damage score of 3.5 (range 3-5) whereas the CO2 pneumoperitoneum group had an average damage score of 1.2 (range 0-3). The damage score of the CO2 pneumoperitoneum group was significantly lower (p⬍0.05). Furthermore, both the average and total weight gain was greater in the CO2 pneumoperitoneum group than the anesthesia only group. Conclusion: This is the first and only such study to describe the potential use of the CO2 pneumoperitoneum as a means of therapy for Crohns-like inflammatory bowel disease. Our results also suggest a better weight gain following administration of CO2 pneumoperitoneum which is an important factor for Crohns patients. Finally, these data suggest that CO2 pneumoperitoneum has important biological tissue effects which significantly influence the response to chronic inflammation. 253. HB-EGF INDUCES FOCAL ADHESION KINASE VIA ERBB-1/ SRC ACTIVATION IN NON-TRANSFORMED

255

RAT INTESTINAL EPITHELIAL (RIE-1) CELLS. O. N. El-Assal, G. E. Besner; Children’s Hospital, Children’s Research Institute, Columbus, OH. Introduction: We have previously demonstrated that endogenous heparin-binding EGF-like growth factor (HB-EGF) is essential for intrinsic restitution and cell migration in vitro, and that administration of recombinant HB-EGF (rHB-EGF) enhances intestinal restitution in rats subjected to intestinal ischemia/ reperfusion injury in vivo. HB-EGF-induced restitution requires activation of the EGF receptor (ErbB-1) and the MEK/ERK and PI3K/Akt signaling pathways. Focal adhesion kinase (FAK) is involved in cell adhesion, migration, and survival. In a continued effort to identify the molecular mechanisms of HB-EGF-induced cell migration and restitution, we now demonstrate for the first time that HB-EGF induces activation of FAK/Paxillin molecules. Methods: RIE-1 cells were serum starved for 6h and then treated with HB-EGF (10 ng/ml) for various time intervals. The specific inhibitors AG1478, PP2, and U0126 were used to specifically block ErbB-1, Src, and MEK/ERK kinase activity, respectively. When inhibitors were used they were added 30 min prior to HB-EGF addition. Cell lysates were prepared and proteins separated on SDS/PAGE gels. Activation of ErbB-1, FAK, ERK, and Paxillin was determined using phosphospecific antibodies. Results: HB-EGF induced ErbB-1 receptor and FAK activation as early as 2 minutes after its addition. Activation of FAK was followed by activation of Paxillin, an immediate FAK substrate , indicating the potency of activated FAK. Blocking of ErbB-1 receptor activity using AG1478 abolished HB-EGF-induced FAK phosphorylation as well as the subsequent activation of Paxillin. Since Src kinase is involved in FAK activation, and in some biological systems activated FAK induces Src activation, we next investigated the effect of Src inhibition on HB-EGF-induced FAK/Paxillin activation. Inhibition of Src activity using PP2 resulted in a dose-dependent inhibition of HB-EGFinduced FAK and Paxillin activation. HB-EGF also induced MEK/ ERK pathway activation. Since Src is involved in ERK activation in some biological systems, we evaluated the role of Src in HB-EGFinduced MEK/ERK activation in RIE-1 cells. Blocking of Src by PP2 slightly, but not significantly, reduced HB-EGF-induced ERK activation, indicating that HB-EGF induces MEK/ERK activation via multiple mechanisms. Conclusion: HB-EGF induces activation of FAK/Paxillin via activation of the ErbB-1 receptor and requires Src activation. HB-EGF-mediated activation of FAK/Paxillin demonstrates that the potent chemotactic effects of HB-EGF on intestinal epithelial cells are mediated via activation of multiple signaling pathways involved in cell motility and adhesion.

ONCOLOGY I- Tumor Biology 1 254. IN VIVO ACTIVATION OF RAF-1 INHIBITS TUMOR GROWTH AND DEVELOPMENT IN A XENOGRAFT MODEL OF HUMAN MTC. Vaccaro AM, Chen H, Kunnimalaiyaan M; University of Wisconsin Medical School Background: Besides surgical resection, there are no effective therapies for medullary thyroid cancer (MTC), a neuroendocrine (NE) tumor derived from parafollicular C-cells. We have previously shown that activation of the raf-1/MEK/ERK1/2 pathway in MTC cells suppresses tumor cell growth and calcitonin secretion in vitro, suggesting that manipulation of this signaling pathway could be a strategy to treat MTC. However, the in vivo effects of raf-1 activation in MTC tumors have not been characterized. Methods: To study the effects of raf-1 activation in vivo, we utilized a murine model of MTC. Human MTC TT cells were transduced with an estrogen (E2)inducible raf-1 construct creating TT-raf cells. TT or TT-raf cells (10 6) were injected into the flank of nude athymic mice. In the first experiment, 56 mice were divided into 4 groups (see Table). To determine if raf-1 activation would prevent MTC tumor formation, E2 or vehicle sustained release pellets were implanted at the time of

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

tumor cell injection. Mice were sacrificed after 20 days. To determine if raf-1 activation could inhibit growth of established tumors, E2 and control pellets were implanted after tumor development in 20 mice (Experiment #2). Activation of raf-1 in E2 treated mice was confirmed by Western blot for phosphorylated ERK1/2 in the MTC tumors. Results: In experiment #1, mice with TT tumors treated with control and E2 pellets and mice with TT-raf tumors treated with control pellets had a high rate of MTC tumor development. However, activation of raf-1 by E2 pellet treatment in mice with TT-raf tumors led to a significant decrease in MTC tumor formation. Similarly, in experiment #2, activation of raf-1 in Group 4 mice suppressed MTC tumor growth. Activation of raf-1 in mice in Group 4 for both experiments was confirmed by the presence of phosphorylated ERK1/2 in the MTC tumors. Conclusion: In vivo activation of raf-1 in a murine model of MTC led to a reduction in MTC tumor development and inhibited the growth of established MTC tumors. These results suggest that the strategies to activate the raf-1/MEK/ERK1/2 signaling pathway may be a viable approach to treat patients with unresectable MTC.

Group

Cells

Pellet implanted

1 2 3 4 p-value

TT TT TT-raf TT-raf —

Control E2 Control E2 —

Experiment #1 No. animals with MTC tumors

Experiment #2 MTC tumor volume (mm 3)

11/14 12/14 12/14 2/14 ⬍0.05

4.0 ⫾ 0.7 8.1 ⫾ 0.8 3.2 ⫾ 0.5 0.6 ⫾ 0.4 ⬍0.05

Chi-squared, ANOVA 255. TGF-BETA SIGNALING PATHWAY INACTIVATION AND CELL CYCLE DEREGULATION IN HUMAN HEPATOCELLULAR CANCER CELL LINES AND ELFⴙ/ⴚ TISSUES. K. Kitisin, E. Volpe, S. S. Kim, W. Jogunoori, Y. Tang, V. Katuri, K. Shetty, B. Mishra, L. Mishra, L. Johnson; Georgetown University, Washington, DC. Hepatocellular cancer (HCC) is increasing in the United States and is the fifth most common solid malignancy worldwide. Nearly 500,000 cases of HCC are diagnosed each year with a mortality rate equivalent to its incidence. Studies on human HCCs reveal various molecular events that may play crucial roles in human hepatocarcinogenesis, specifically in the transforming growth factor-beta (TGF␤Beta) signaling pathway. As a potent growth-inhibitory cytokine, TGF-␤ Beta is believed to be important in the maintenance of hepatic tissue homeostasis. TGF-␤ -Beta can induce antiproliferative gene responses at any point during the cell division cycle, though the most effective inhibition of cell cycle progression occurs during G1. Arrested cells in the G1 phase display significant downregulation in expression of Cdk2, Cdk4, cyclin D2, cyclin D3 and cyclin A _ escape from this response is a hallmark of many cancer cells. It has also been shown that Cdc2 and Cdk2 are activated in human HCC. An important modulator of the TGF-␤ Beta signaling pathway is an adaptor ␤Beta-spectrin embryonic liver fodrin (ELF) protein. Our lab has demonstrated that as many as 35% of elf⫹/⫺ mice (7/20) develop HCC and showcase phenotypic changes such as increased centrilobular steatosis, dysplasia, and high grade atypia. Aims: 1. To determine whether TGF-␤ -Beta signaling proteins/ELF are inactivated in human HCC cell lines 2. To analyze cell cycle disruption in human HCC and determine the correlation of this disruption in relation to the TGF-␤ -Beta signaling pathway. Methods and Results: 1. Expression of ELF and other proteins involved in the TGF-␤ -Beta signaling pathway, particularly Smad2, Smad4, TGF-␤ -Beta receptor I, TGF-␤ -Beta receptor II, and PRAJA, were examined in human HCC cell lines SNU-182, SNU-398, SNU-449, SNU-475, and HepG2. ELF

expression was lost in one human HCC cell line (SNU-398), and decreased in another (SNU-475). Smad2 expression was lost in all five HCC cell lines, and TGF-␤ -Beta receptor II in three cell lines (SNU-398, SNU-182, and SNU-475). 2. Markedly elevated expression of c-myc and phosphorylated myc was seen in the elf⫹/⫺ cancers. Immortalized mouse embryonic fibroblasts (MEFs) derived from elf-/- mice also demonstrated an increase in cdk4 levels. TGF-␤ -Beta treatment of hepatocytes and wild type MEFs resulted in a marked suppression of myc and hTERT that was dose dependent. 3. Null MEFs displayed increased Cdk4-kinase activity resulting in hyperphosphorylation of all three members of the Rb-family, pRb, p107, and p130. 4. Immunohistochemical labeling of elf⫹/⫺ HCC tissue revealed markedly increased ras, cyclin D1, MDM2, phosphorylated Rb, and cdk4. Conclusion: Diminished or absent ELF expression in mouse and human HCC as well as cell cycle deregulation suggest that the disruption of TGF-␤ -Beta and the loss of ELF can serve as a primary event in progression towards a fully transformed phenotype. Patients with HCC are faced with a poor prognosis despite treatment through liver resections or liver transplantation. Tumor recurrence is common and poses poor clinical outcome. Therefore, exploration of the mechanisms behind inactivation of the TGF-␤ -Beta signaling pathway and its restoration hold promise for new therapeutic approaches in gastrointestinal cancers. 256. APOPTOSIS IN A MALIGNANT CELL LINE INDUCED BY A NOVEL SUICIDE GENE THERAPY EXPLOITING ELEVATED LEVELS OF EUKARYOTIC INITIATION FACTOR 4E. Byrnes KW, Li B, Li J, Turnage R, Chu Q; LSU-Health Sciences Center in Shreveport. Background: Eukaryotic Initiation Factor 4E (eIF4E), facilitates the translation of mRNAs with long 5’ un-translated regions (UTRs) and thus regulates protein synthesis. This protein has been found in elevated quantities in breast, colon, and head and neck cancers. To exploit this dysfunction, the 619 base pair 5’UTR of FGF-2 was spliced upstream of the herpes simplex virus thymidine kinase gene in an adenovirus vector (Ad-HSV-UTK), with the expectation that TK will be expressed in cells which overexpress eIF4E, and thus yield these cells susceptible to gancyclovir. In this study, we investigated the in vitro activity of this suicide gene therapy regimen against the rat small bowel adenocarcinoma cell line CRL 1677. Methods: One group was transfected with the Ad-HSV-UTK vector while one group (the control arm) was not transfected. 24 hours after transfection each group was further divided into 5 groups, each receiving a progressive dose of gancyclovir: group 1⫽0, group 2⫽10mcg/ml, group 3⫽50 mcg/ml, 4⫽100 mcg/ml, 5⫽1000 mcg/ml. Successful transfection of the cell line was determined by Western Blot analysis for the gene product thymidine kinase. Cell viability was assessed by the MTT assay. Induction of apoptosis was determined by flow cytometry using the Tunnel assay. Results: Western Blot analysis confirmed successful transfection of the cell line. Marked cytotoxicity was noted by the MTT assay in the transfected group with a 100-fold less concentration of gancyclovir as compared to the control groups, which received gancyclovir alone. Flow cytometric analysis for Annexin V-FITC revealed early events of apoptosis in each transfected group after treatment with gancyclovir (with a peak at the 50 mcg/ml group). Early events of apoptosis were not evident in any of the control arms. Conclusion: By splicing a 5’UTR upstream of HSV-TK, thymidine kinase expression was detected in the CRL 1677 cell line. Marked cytotoxicity was evident in the treatment groups, even at a much lower concentration of gancyclovir. Early events of apoptosis were also evident in the treatment group. Suicide gene therapy targeting the overexpression of eIF4E is effective in inducing apoptosis and cell death in this malignant cell line. 257. VASCULAR ENDOTHELIALGROWTH FACTOR (VEGF) INHIBITION CAUSES AN INCREASE IN PERICYTES AND PLATELET DERIVED GROWTH FACTOR RECEP-