The APC tumor suppressor regulates apoptosis and proliferation in human breast cancer cells

262

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

have been implicated in the malignant phenotype of a range of cancers. More specifically, overexpression of HMG A1 has been associated with tumor metastasis, with very low/absent expression being present in normal tissues. It remains controversial if HMG A1 is metastasis-inducing or -associated. HMG A1 proteins (HMGA1a and HMGA1b) represent two alternatively-spliced proteins which differ by 11 amino acids and are encoded by the same gene. HMG A1 proteins are non-histone nuclear proteins, which bind the AT-rich regions in the minor groove of DNA via three AT-hook domains. They play an important role in regulating gene expression by acting as architectural nuclear proteins. Hypothesis: We hypothesize that HMG A1 is a determinant of pancreatic adenocarcinoma cellular invasiveness. Methods: We studied pancreatic adenocarcinoma cell lines, MiaPaCa2 (low/moderate expression of HMG A1) and PANC1 (overexpression of HMG A1). Short-hairpin RNA (shHMGA1) expression vectors targeting all transcriptional variants of HMG A1 gene were used. Four shHMGA1 constructs with different target sequences were evaluated for degree of HMGA1 knockdown and construct with highest degree of knockdown was chosen for further evaluation. Pooled stable transfectants of short-hairpin RNA expressing cell lines were developed following chemical transfection and antibiotic selection. Cellular invasiveness was quantified using modified Boyden chamber assay. Knock-down of HMG A1 expression was confirmed using Western blot analysis. Results: We achieved 55-65% silencing of HMG A1 protein expression when compared to control group (shRNA targeting luciferase) [relative density (normalized to actin): shHMG A1 vs. shControl; MiaPaca2: 1.00⫾0.3 vs. 2.2⫾0.3 arbitrary units, PANC1: 1.0⫾0.5 vs. 2.8⫾0.6, both cell lines p⬍0.05]. shHMG A1 transfected MiaPaCa2 cells showed significantly lower invasive capacity compared to controls (relative invasion:0.4⫾0.3 vs. 2.2⫾0.7 arbitrary units,p⫽0.03). Similarly, shHMGA1 reduces invasion of PANC1 cell lines (relative invasion: 1.0⫾0.2 vs. 3.0⫾0.8, p⫽0.03). There was no significant effect on cellular migration. Additional studies suggest that matrix metalloproteinase 9 and Akt are downstream mediators of HMG A1. Conclusion: HMG A1 plays a role in inducing tumor invasion and represents a novel therapeutic target for pancreatic cancer, warranting further investigation. 271. CARCINOID TUMOR CELL GROWTH SUPPRESSION BY PI3 KINASE-AKT PATHWAY INHIBITION. M. Kunnimalaiyaan, M. Kloosterboer, H. Chen; University of Wisconsin, Madison, WI. Introduction: The PI3 Kinase-Akt signaling pathway is known to promote tumor cell survival by suppressing pro-apoptotic proteins. We have previously shown that human carcinoid tumor cells utilize several signal transduction pathways to modulate cellular growth and neuroendocrine (NE) marker production. Furthermore, human carcinoid cells have endogenously high levels of active, phosphorylated Akt. Therefore, we hypothesized that the inhibition of the PI3 Kinase-Akt pathway may lead to a reduction of carcinoid tumor cell growth and NE marker production. Methods: Human gastrointestinal (GI) carcinoid BON cells were treated with varying concentrations of the PI3 kinase inhibitor LY294002 (0-100 ␮M) for a two-day time period. Western analyses were performed for active, phosphoAkt, total Akt, and the NE marker human achaete-scute homolog1 (hASH1). Cellular growth was measured both by MTT assay and cell counts for 8 days. Results: Treatment of BON cells with LY294002 led to a dose-dependent reduction in the levels of the active, phosphorylated Akt, illustrating the successful inhibition of the PI3 Kinase-Akt pathway. Moreover, LY294002 significantly suppressed carcinoid tumor cell growth and production of hASH1. Importantly, these reductions in cellular growth and NE marker production were directly proportional to the degree of Akt inhibition. Conclusion: Human carcinoid tumor cell growth and hASH1 production appears to be dependent upon activation of the PI3 Kinase-Akt signaling pathway. Therefore, inhibition of this important signal transduction

pathway could be a potential therapeutic strategy to treat and palliate patients with carcinoid tumors.

ONCOLOGY III-Tumor Biology 3 272. EFFECTS OF THE MEK INHIBITOR UO126 ON GROWTH OF CANCER CELL LINES HEPG2, MIAPACA, PANC-1, AND HT-29. Mackin KM, Deming DA, Geiger P, Chen H, Holen K; University of Wisconsin Madison Medical School Introduction: The Ras/Raf/Mek/Erk intracellular signaling pathway connects cell surface receptors to gene transcription factors which regulate gene expression to control cellular proliferation, cell differentiation, and apoptosis. This pathway is erroneously activated in many types of cancer, and is thought to play a crucial role in tumorogenesis. For that reason, this pathway has been of great interest as a target for the development of anti-neoplastic agents. UO126 (Cell Signaling Technology) has been shown to inhibit Mek, a critical component of the MAP kinase cascade. We therefore hypothesize that by treating tumor cell lines with UO126, we can block this pathway, leading to a decrease in tumor growth. Methods: The cancer cell lines HepG2 (hepatoma), Ht-29 (colon), MiaPaca (pancreas) and Panc-1 (pancreas), were cultured in Dulbeco’s modified eagles medium (Gibco, Grand Island, NY) supplemented with fetal bovine serum, penicillin, and streptomycin. The cells were treated with UO126 for 45 minutes at concentrations ranging from 20-50 uM. An MTT assay, which uses absorbance to measure growth, was performed to determine the effect of UO126 on cellular proliferation. To evaluate Mek activity, western blotting was used to analyze pErk both before and after the addition of UO126. In cell lines that did not show significant baseline pErk activity, we activated the cascade with ZM336372, a ras activating agent, in order to show that UO126 lead to a decrease in pErk even when ZM336372 was present. Results: Our experiments showed a significant decrease in the pErk levels after treatment with UO126. We also showed a significant decrease in cell growth in all four cell lines after treatment with UO126. Conclusion: UO126, a Mek inhibitor, showed considerable activity in the cell lines tested and further studies in other tumor cell lines are warranted. 273. THE APC TUMOR SUPPRESSOR REGULATES APOPTOSIS AND PROLIFERATION IN HUMAN BREAST CANCER CELLS. B. J. Boulton, M. B. Ebetino, T. A. Willson, K. H. Goss; University of Cincinnati College of Medicine, Cincinnati, OH. Introduction: Alteration of the APC tumor suppressor gene is the most common genetic alteration in sporadic colorectal cancer and is observed in as many as 45% of sporadic human breast cancers. Studies performed in colorectal cancer cells suggest that APC is a multifunctional protein that likely acts as a tumor suppressor by inhibiting cell proliferation and inducing apoptosis. Furthermore, aberrant activation of the ␤-catenin/Wnt signaling pathway, as a direct result of APC inactivation, has been associated with both of these processes. However, the role of APC in mammary cell regulation and breast cancer tumorigenesis remains unknown. We hypothesize that APC regulates normal mammary epithelial cell homeostasis, perhaps by its ability to negatively regulate the ␤-catenin/Wnt pathway, and loss of this regulation by APC is important in breast tumor formation. Methods: In this study, we overexpressed human APC tagged with green fluorescent protein (GFP) in a nontransformed mouse mammary epithelial cell line, EpH4, as well as a human breast cancer cell line, MDA-MB-231. After transfection with APC-GFP or GFP alone and a 36-hour incubation, the percentage of cells undergoing apoptosis was analyzed using immunofluorescent detection of a caspase cleaved epitope of cytokeratin 18 that is uniquely found during early apoptosis. Cell proliferation assays were

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS performed by immunofluorescent labeling of bromo-deoxyuridine (BrdU) 24 hours after introduction into cells transfected with APCGFP or GFP alone. We next examined a key component of the Wnt signaling pathway, ␤-catenin, by immunofluorescence and western blotting in cells that overexpressed APC or GFP alone. Results: APC overexpression resulted in a statistically significant (p⬍0.01) increase in apoptosis in both the normal (64% vs. 14%) and transformed (80% vs. 14%) mammary cell lines when compared to cells transfected with GFP alone. There was a statistically significant (p⬍0.01) reduction in BrdU incorporation in APC-expressing breast cancer cells; 36% of MDA-MB-231 cells overexpressing APC demonstrated BrdU uptake as compared to 63% of cells transfected with only GFP. No difference in cell proliferation, however, was observed with APC introduction into non-transformed mammary cells when compared to GFP (62% vs. 64%). In analyzing the activation of the ␤-catenin/Wnt pathway, EpH4 cells overexpressing APC demonstrated dramatically reduced expression of ␤-catenin, while the reduction of ␤-catenin in APC-GFP transfected breast cancer cells was less obvious. Conclusion: Taken together, our results demonstrate that APC plays a vital role in the regulation of apoptosis in both normal and transformed mammary epithelial cells and that APC expression is sufficient to inhibit cell cycle progression in breast cancer cells, although the mechanism by which APC exerts these effects may be independent of ␤-catenin/Wnt pathway regulation. These data suggest that the APC tumor suppressor may play distinct molecular roles in breast and colorectal cancer tumorigenesis, a finding that could have important clinical and therapeutic applications. 274. ETHANOL-TGF-␣-MEK GROWTH SIGNALING MAY PROMOTE ALCOHOL ASSOCIATED HUMAN HEPATOCELLULAR CARCINOMA. M. Hennig 1, P. Klein 1, J. Matos 1, A. Norris 1, S. Noble 1, C. Doyle 1, I. McKillop 2, E. Wiebke 1, C. M. Schmidt 1; 1IU School of Medicine, Indianapolis, IN, 2University of North Carolina, Charlotte, Charlotte, NC. The incidence of alcohol associated hepatocellular carcinoma (HCC) is rapidly rising but the molecular pathogenesis of this relationship is unclear. Alcohol’s effects on human transforming growth factor alpha (TGF-␣), a molecule sufficient to induce HCC in TGF-␣ transgenic mice, have not been examined. Human HCC cell lines (HepG2, SKHep) were treated with clinically relevant concentrations of ethanol (10 - 40 mM) for time points ranging from 30 minutes to 24 hours. Ethanol and TGF-␣ levels were quantified by ELISA. TGF-␣ signaling was manipulated with TGF-␣ neutralization antibody, U0126 (MEK inhibitor) and LY (PI3 Kinase inhibitor). Growth was determined by trypan blue excluded cell counts. Protein expression of P-Erk, total ERK, P-AKT and total AKT was determined by Western blot. TGF-␣ levels in HepG2 cells increased (166 - 424 %) after 24 hours of ethanol (10 - 40 mM) treatment. Similarly, TGF-␣ levels in SKHep cells increased (112 - 177 %) after 24 hours of ethanol (10 - 40 mM) treatment. Correspondingly, cell counts increased in HepG2 (149 ⫾ 12 %, P⬍0.05) and SKHep (174 ⫾ 29 %, P⬍0.05) in response to ethanol (10 - 40 mM) treatment. TGF-␣ neutralization antibody (0.25 ng/mL) effectively abrogated this increase in cell counts in HepG2 (102 ⫾ 11 %, P⫽NS) and SKHep (92 ⫾ 7 %, P⫽NS) cells. Exogenous administration of recombinant TGF-␣ at levels corresponding to ethanol induced levels of TGF-␣ mimicked the growth induced by ethanol (151 ⫾ 18 % HepG2, 140 ⫾ 17 % SKHep, P⬍0.05). Although no changes were observed in total ERK, P-Erk levels increased early at 30 minutes and 3 hours with ethanol treatment in both HepG2 and SKHep cells. A similar increase in P-Erk was demonstrated with exogenous TGF-␣ treatment at 30 minutes and 3 hours in both HepG2 and SKHep cells. P-Erk increases were blocked with U0126 (0.1 ␮M). Correspondingly, U0126 suppressed exogenous ethanol-induced and TGF-␣-induced growth increases. No changes were seen in P-Akt or total Akt with ethanol or exogenous TGF-␣ suggesting no role for Ethanol-TGF-␣- Pi3 Ki-

263

nase survival signaling. In conclusion, our results show that ethanol up-regulates TGF-␣ levels in HCC cells and ethanol-TGF-␣ signaling enhances HCC growth likely through Mek independent of Akt. Previous studies demonstrate no ethanol-TGF-␣-Mek signaling in normal hepatocytes. Thus, we hypothesize that ethanol promotion of HCC may depend upon initiated hepatocytes acquiring this cancer specific signaling. 275. HYPOXIA MEDIATES METASTASIS AND INVASION OF BREAST CANCER CELLS VIA THE CHEMOKINE RECEPTOR CXCR4. E. M. Faul, H. P. Redmond, J. H. Wang; Cork University Hospital, Cork, IRELAND. Aims: Experimental evidence has demonstrated that hypoxia stimulates tumor progression and resistance to both chemotherapy and radiotherapy. Recently, the chemokine receptor CXCR4 has been implicated in organ-specific metastasis of breast cancer, metastatic cancer cells simply co-opting signals that normally control lekocyte transport. We hypothesized that hypoxia would increase CXCR4 expression via HIF-1alpha (hypoxia inducible factor 1alpha) and thus stimulate increased metastasis and invasion of breast cancer cells. We also compared Her 2 positive and Her 2 negative cell lines to determine if a difference in their response to hypoxia might account for the increased aggression of Her 2 positive breast cancer. Methods: Four cell lines were cultured; SKBR3, MCF-7, BT474 and MDA MB 231. These cell lines were then subjected to hypoxia for 6, 12, 24 or 48 hrs. Total RNA was extracted for RT-PCR. Protein was extracted for western blots. Chemoinvasion was verified using a modified Boyden chamber. Results: Hypoxia stimulated increased CXCR4 expression at both RNA and protein levels. The increase in CXCR4 expression in Her 2 positive cells was more pronounced with up to 35-fold increases in CXCR4 expression after hypoxia in one Her 2 positive cell line. The increase in CXCR4 expression translated to enhanced chemoinvasion of breast cancer cells. Conclusion: Tumour hypoxia significantly increases CXCR4 expression in breast cancer causing increased invasion and metastasis of cells. The increased aggression of Her-2-neu positive breast cancer may be due to increases in CXCR4 expression stimulated by hypoxia. These findings identify a novel therapeutic target in breast cancer treatment. 276. RESTORATION OF E-CADHERIN AND APOPTOSIS OF HUMAN PANCREATIC CANCER CELLS AFTER RNA INHIBITION OF MUTANT K-RAS. J. Fleming, G. Shen, R. Brekken; University of Texas Southwestern Medical Center, Dallas, TX. Background: We have demonstrated that RNA interference (RNAi) against mutant K-ras inhibits growth of pancreatic cancer cells: however, not all cell lines are equally sensitive. In epithelial cells the adherens junction protein E-cadherin protects against apoptosis and loss E-cadherin occurs during Ras-mediated transformation. We hypothesize that changes in expression of E-cadherin is coincident with apoptosis after mutant K-ras RNAi in pancreatic cancer cells. Methods: RNA oligonucleotides specific for activating K-ras mutations in two human pancreatic cancer cell lines, Panc-1 and MiaPaca-2, were synthesized and duplexed. Control oligonucleotides were also generated. Pancreatic cancer cells were maintained in culture then seeded and exposed to mutant specific K-ras or control siRNA (25, 50 or 100 nm) for six hours. At 24, 48 and 72 hours cells were harvested for analysis of protein (Western blot) or cells were fixed and protein expression examined by immunocytochemistry (ICC). Using these same timepoints cell survival, growth, and migration was assessed by; bioreduction of a MTS tetrazolium salt at 490 nm (MTS), fluorescent microscopy for nuclear condensation (Hoescht nuclear staining). Results: Cell lysates from Panc-1 and MiaPaca-2 cells both demonstrated reduction in mutant K-ras protein 24 hours after exposure to 25 nm of mutant specific but not control siRNA. Neither cell line expressed E-cadherin at baseline