resistance in advanced breast cancers. Methods: Immunofluores-cent staining for DACH1 was performed on both normal breasttissue and breast cancer tissue to look for differential expression,with analysis by the AQUA/PM2000 platform. Proteomic analysiswas used to identify DACH1 binding proteins. PELP1 serves as anER� cointegrator protein. Mass spectrometry and sequence anal-ysis identified PELP1 as a DACH1 binding protein. Immunofluo-rescent staining was also performed on the cellular level, analyz-ing the MCF-7 breast cancer cell line by transiently transfectingwith the DACH1 and PELP1 expression vectors, treating witheither estradiol or vehicle, and examining for co-localization. Lu-ciferase assays were performed in MCF-7 cells with the estrogenreceptor-alpha response element and co-transfection with DACH1and PELP1. Immunoprecipitation was performed to check fordirect binding of DACH1 to PELP1. Results: Analysis of immu-nofluorescent staining of breast tissue revealed that in normaltissue DACH1 is localized to the nucleolus, while in canceroustissue it can be found throughout the cell. On the cellular level,with immunofluorescence DACH1 was found to be localized to thenucleus when transiently transfected into MCF-7 cells, co-localizing with PELP1, but the addition of estradiol causedDACH1 to dissipate throughout the cytoplasm, while PELP1 re-mained in the nucleus. Luciferase assays using the ER responseelement in breast cancer cells, showed that the addition of in-creasing amounts of DACH1 led to decreased estradiol-inducedactivation of the ER reporter. Immunoprecipitation revealed thatDACH1 binds directly to PELP1, and requires the carboxyl ter-minus, as use of a truncation mutant form of DACH1 abolishedbinding to PELP1. Conclusions: The cell fate determination fac-tor DACH1 directly binds to the co-regulator PELP1, repressingestrogen receptor activity. Addition of estradiol to breast cancercells leads to translocation of DACH1 from the nucleus to thecytoplasm, and increased estrogen receptor activity.

139. SILENCING ANGIOCIDIN EXPRESSION REDUCES TU-MORIGENICITY OF COLON CANCER CELLS. NeetiAgarwal, Catherine Liebig, Jonathan A. Wilks, Daniel Albo;Baylor College of Medicine, Houston, TX

Introduction: Angiocidin is over-expressed in colon cancer. Re-cent antibody and inhibitory peptide data suggest a tumorigenicactivity for angiocidin. We now hypothesize that silencing angio-cidin expression using specific siRNA would lead to a decrease intumorigenic properties of colon cancer cells. Methods: We use thehigh-angiocidin expressing KM12L4 colon cancer cell line. Usingretroviral transduction we generated stable Angiocidin siRNAKM12L4 clones expressing a specific siRNA against angiocidin(L4-Angiosi1, L4-Angiosi2 cells). Controls included KM12L4 cellstransduced with vector alone (L4-V) or a non-specific siRNAagainst GFP (L4-GFPsi). Angiocidin protein and mRNA expres-sion were determined by Western blot and quantitative RT-PCR,respectively. Tumor cell proliferation was determined by MTTassay. Tumorigenicity was evaluated in a subcutaneous injectionnude mouse model. Tumor sizes were measured every third day.Statistical analysis was performed by ANOVA and student’st-test. Results: Angiocidin siRNA transfection achieved a 70-90%reduction in angiocidin mRNA and protein expression in our L4-Angiosi1 and L4-Angiosi2 cells compared to wild type and controlcolon cancer cells (Fig.1; Fig.2; p�0.006). L4-Angiosi1 and L4-Angiosi2 cells exhibited 2 to 3-fold lower proliferation rates thanwild type and control colon cancer cells (Fig.3; p�0.001). Nudemice injected with angiocidin siRNA colon cancer cells developedsignificantly smaller tumors compared to control animals (meantumor volumes of 230 mm3 vs 971 mm3, respectively, Fig.4;p�0.02). Conclusion: Our data shows that angiocidin siRNA in-hibition reduces tumorigenicity of colon cancer cells.

237ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

140. PANCREATIC CANCER STEM CELLS ARE RESISTANTTO IONIZING RADIATION AND THE CHEMOTHERA-PEUTIC AGENT GEMCITABINE. Cheong J. Lee, Diane M.Simeone, Chenwei Lee, Steve Zielske, Joseph Dosch, MatsLjungman; University of Michigan, Ann Arbor, MI

Background: Even though major advancements have been madein the management of many cancers, the prognosis for pancreaticcancer still remains dismal. A key characteristic of pancreaticcancer that makes this disease so lethal is its resistance to ioniz-ing radiation and chemotherapy. Recent studies have suggestedthat tumor initiating “cancer stem cells” in breast and braincancers may be responsible for conferring the property of chemo-and radio-resistance in solid tumors. We have recently identifiedhuman pancreatic cancer stem cells that express the cell surfacemarkers CD44�CD24�ESA� (0.2-0.8% of all pancreatic cancercells). We hypothesize that pancreatic cancer stem cells are resis-tant to standard therapies used to treat this disease. Methods:Three samples of human pancreatic adenocarcinoma obtainedfollowing surgical resection were established as xenografts inimmunocompromised NOD-SCID mice. All xenograft experimentswere performed in triplicate. Xenografts approximately 1 cm2 indiameter were irradiated either with a single dose of 8 Gy or fourfractionated doses of 2.5 Gy for a total of 10Gy. In separateexperiments, mice bearing primary pancreatic cancer xenograftswere treated with a 125mg/kg bi-weekly dose of gemcitabine for 4weeks. The population of CD24�CD44�ESA� cells in treated andcontrol tumors was assessed by immunofluorescent staining andflow cytometry. TUNEL assays were performed on tissue sectionsof xenografts following ionizing radiation or chemotherapy treat-ment to assess the extent of apoptosis. Tumorigenicity of treatedand control sorted cells was assessed following in vivoimplantation. Results: The CD24�CD44�ESA� subpopulation ofpancreatic tumor cells increased 5.8 � 1.7 fold (p�0.05) followinga single radiation dose of 8 Gy on pancreatic adenocarcinomaxenografts (n � 3 separate experiments). Administration of amore clinically relevant fractionated radiation dose of 2.5 Gy � 4increased the cancer stem cell population 4.25 �1.3 fold (p � 0.05,n � 3). Irradiation did not induce CD24 or CD44 expression inmarker negative tumor cells. The greatest percentage increase inCD24�CD44�ESA� cells occurred 48 hours to 1 week followingirradiation, the same time points where the highest rates of apo-ptosis occurred. CD24�CD44�ESA� cell populations returned tobasal levels (0.2-1% of tumor cells) by 3 weeks following treat-

ment, suggesting that this enhanced cancer stem cell populationmay undergo differentiation. Of functional relevance, single cellsuspensions of cancer cells from irradiated tumors showed en-hanced tumorigenicity when implanted into NOD-SCID mice com-pared to non-irradiated cells, suggesting that the enriched cancerstem cell population following irradiation may enhance tumorige-nicity. Similar results were obtained in animals treated withgemcitabine. Conclusion: These data suggest that human pan-creatic cancer stem cells are resistant to irradiation and chemo-therapy. These findings have significant implications for treat-ment strategies of pancreatic cancer.

141. AKT2 IS A CRITICAL REGULATOR OF COLORECTALCANCER METASTASIS ESTABLISHMENT. Piotr G.Rychahou, JungHee Kang, Prateek Gulhati, L. Andy Chen, DaiH. Chung, B. Mark Evers; The University of Texas MedicalBranch, Galveston, TX

Introduction: Colorectal cancer (CRC) is the second leadingcause of cancer deaths in the US; approximately 35-55% of pa-tients with CRC will develop liver metastases during the course oftheir disease. Previously, we demonstrated the importance of thephosphatidylinositol 3-kinase (PI3K) pathway, acting through itsdownstream effector protein, Akt, in the proliferation and metas-tasis of CRC using novel preclinical models of liver metastasis.The purpose of our present study was to delineate the roles of theAkt isoforms (Akt1 and Akt2) in the systemic invasion and me-tastasis of CRC. Methods: (i) First, we evaluated the relativeexpression of Akt1 and Akt2 in different histotypes of human CRCand representative normal tissues (n � 45) using tissue microar-ray analysis and, in experimental colon neoplasms, by immuno-histochemistry. To develop additional supportive evidence for arole for Akt2 in CRC metastasis, we compared expression betweenprimary CRC and matched metastases in the same patients (n �36) by tissue microarray analysis. The relative levels of Akt2mRNA expression were examined in different stages of CRC andnormal tissue (n�48) using real time PCR. (ii) To further analyzethe contribution of the Akt isoforms to CRC metastasis, we tran-siently expressed Akt1 or Akt2 in non-metastatic human CRCcells (ie, SW480GFP and Caco-2GFP); the cells were injected into thespleens of athymic nude mice and mice were examined periodi-cally by whole-body, real-time fluorescence imaging for in vivovisualization of metastasis. Conversely, we transfected a meta-static CRC cell line (ie, KM20GFP) with siRNA to either Akt1 orAkt2 or non-targeting control (NTC); cells were injected in thespleens of athymic nude mice and metastasis assessed as de-scribed above. Results: (i) Marked immunoreactivity was detectedin premalignant colonic mucosa, 70% of experimental murinecolon neoplasms and in different histotypes of human CRC (well-or moderately-differentiated); Akt1 expression was lower thanAkt2 expression for all histotypes. In 32 of 36 cases, Akt2 expres-sion was greater in the liver metastases than in primary colontumors. In contrast to the higher expression of Akt2 in many ofthe liver metastases, there was no differential pattern of Akt1expression between primary tumor and liver metastasis. (ii) Sup-pression of Akt2 expression in the highly metastatic KM20 CRCcells specifically inhibited their ability to metastasize in an exper-imental liver metastasis model (see figure comparing Akt2 siRNAvs. control). Overexpression of wild-type Akt1 did not restoremetastatic potential in cells with downregulated Akt2. In con-trast, ectopic expression of wild-type or constitutively active Akt2resulted in metastasis by the non-metastatic CRC cell lines.

238 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS